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Authors: G-Biosciences
Group: G-Biosciences
Summary: The OmniPrep™ kit isolates high quality genomic DNA from many different species and tissue types including animal, plant, bacteria, yeast, fungi, whole blood, and cells in culture. DNA can be isolated from samples high in polysaccharides or other contaminants that are difficult to remove from the DNA preparations.This protocol is for use with gram-positive bacteria. Please refer to the appropriate protocol depending on your application.

Proper citation: G-Biosciences 2016. OmniPrep™ For High Quality Genomic DNA Extraction From Gram-Positive Bacteria. protocols.io dx.doi.org/10.17504/protocols.io.e57bg9n Copy   

•   Source: Protocols.io

Authors: Gloria Pryhuber, Ravi Misra, Tiru Rangasamy, Thomas Mariani, Gautam Bandyopadhyay, Lisa Rogers, Amanda Howell, Claire Wyman, Heidie Huyck, Siva Solleti
Group: Human Cell Atlas Method Development Community, LungMap2 Consortium, URMC Pryhuber Lab
Summary: Lung MAP HTC - BioRepository for Investigation of Neonatal Diseases of the Lung (BRINDL)702.B.3 Combined SOP and Worksheet HTC_Lung_Tissue_Digestion_0100616rev061220Purpose and Scope of the Procedure or Laboratory Assay1.To dissociate and isolate single cell suspensions from human lung samples to meet the standards and needs of the Human Tissue Core. .justify:after { content: ""; display:inline-block; width: 100%; } 2.This protocol covers manual gross dissociation of pieces of lung tissue (lobes or cubes) followed by enzymatic digestion and freezer storage as mixed populations of filtered and counted cells for which viability has been determined. .justify:after { content: ""; display:inline-block; width: 100%; } 3.A separate protocol details the technique of freezing, thawing and further identifying and sorting these mixed cell populations. .justify:after { content: ""; display:inline-block; width: 100%; } Scientific Principles or Validation of Procedure .justify:after { content: ""; display:inline-block; width: 100%; } 1.Fujino et al. Am J Respir Cell Mol Biol. 2012 Apr;46(4):422-30 .justify:after { content: ""; display:inline-block; width: 100%; } 2.Barkauskas, C. E., Cronce, M. J., Rackley, C. R., Bowie, E. J., Keene, D. R., Stripp, B. R., ... & Hogan, B. L. (2013). Type 2 alveolar cells are stem cells in adult lung. The Journal of clinical investigation, 123(7), 3025. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Gloria Pryhuber, Ravi Misra, Tiru Rangasamy, Thomas Mariani, Gautam Bandyopadhyay, Lisa Rogers, Amanda Howell, Claire Wyman, Heidie Huyck, Siva Solleti 2020. 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820. protocols.io dx.doi.org/10.17504/protocols.io.biz5kf86 Copy   

•   Source: Protocols.io

Authors: Helen Dukes, Elizabeth Ottesen
Group: Ottesen Lab
Summary: Sterilization and post-hatch care of gnotobiotic Periplaneta americana, as adapted from Doll et. al. (see below)Doll J, Trexler P, Reynolds L, Bernard G. 1963. The use of peracetic acid to obtain germfree invertebrate eggs for gnotobiotic studies. Am Midl Nat 69:231-239

Proper citation: Helen Dukes, Elizabeth Ottesen 2018. Gnotobiotic Cockroach Gnotocol. protocols.io dx.doi.org/10.17504/protocols.io.t4ceqsw Copy   

•   Source: Protocols.io

Authors: Nina Dombrowski and Kiley Seitz
Group: ECOGEO
Summary: Provides a short introduction to phylogenetics and how to build a phylogenetic tree from concatenated ribosomal marker proteins. Open this protocol inside the virtual machine (details in 'Start Instructions') for easy copy, paste of commands into the command line terminal window.

Proper citation: Nina Dombrowski and Kiley Seitz 2016. ECOGEO 'Omics Training: 4.4 Phylogenetics. protocols.io dx.doi.org/10.17504/protocols.io.fjabkie Copy   

•   Source: Protocols.io

Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. Eosinophils and CD8+T cells synthesize IL-16. Its principal target is CD4+ T cells. It causes CD4+ T cell chemoattraction. [1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-16 in human serum.. protocols.io dx.doi.org/10.17504/protocols.io.bj3gkqjw Copy   

•   Source: Protocols.io

Authors: Susanna Sabin, James Fellows Yates
Group: WarinnerGroup, MPI-SHH Archaeogenetics
Summary: This protocol describes how to sample dental calculus from skeletal remains for biomolecular analysis. This protocol is particularly recommended for sampling calculus from teeth attached to a jaw bone, skull, or skeleton. The primary use-case is for DNA and proteomic analysis.

Proper citation: Susanna Sabin, James Fellows Yates 2020. Dental Calculus Field-Sampling Protocol (Sabin version). protocols.io dx.doi.org/10.17504/protocols.io.bqecmtaw Copy   

•   Source: Protocols.io

Authors: Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin
Group: GigaScience Press, BGI, GIGA, GigaScience Press
Summary: This protocol is provided by BioNano Genomics and was used in:Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin (2016): Supporting data for 'Comparative optical genome analysis of two Pangolin species Manis pentadactyla and Manis javanica'. GigaScience Database.

Proper citation: Huang Zhihai, Xu Jiang, Xiao Shuiming, Liao Baosheng, Gao Yuan, Zhai Chaochao, Qiu Xiaohui, Xu Wen, Chen Shilin 2016. Irys NLRS DNA labeling and Data collection. protocols.io dx.doi.org/10.17504/protocols.io.gahbsb6 Copy   

•   Source: Protocols.io

Authors: Michael Crone
Summary: MinElute columns can also be used on any vacuum manifold with luer connectors (e.g., QIAvac 24 Plus or QIAvac 6S with Luer Adapters). The following protocol is designed to extract and purify DNA of 70 bp to 4 kb from standard or low-melt agarose gels in TAE or TBE buffer using vacuum-driven processing resulting in high end-concentrations of DNA. Up to 400 mg agarose can be processed per MinElute column.

Proper citation: Michael Crone 2015. Qiagen MinElute Gel Extraction. protocols.io dx.doi.org/10.17504/protocols.io.c2fybm Copy   

•   Source: Protocols.io

Authors: Lucas Armitage, Mark Wallet
Summary: This protocol is a modification of a previously published method by Wilgenburg et al (PLoS One, 2013) to obtain monocytes and Dendritic cells (DCs) from induced Pluripotent Stem Cells (iPSCs) lines. 

Proper citation: Lucas Armitage, Mark Wallet 2018. The production of monocyte-derived dendritic cells from human iPSCs. protocols.io dx.doi.org/10.17504/protocols.io.ng8dbzw Copy   

•   Source: Protocols.io

Authors: Matthew Sullivan Lab
Group: Earth Microbiome Project

Proper citation: Matthew Sullivan Lab 2019. Quanti-iT™ Pico Green dsDNA Assay (Invitrogen P7589). protocols.io dx.doi.org/10.17504/protocols.io.c5zy75 Copy   

•   Source: Protocols.io

Authors: Angel Justiz-Vaillant
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. IL-31 is produced mainly by Th2 cells and dendritic cells. It is a proinflammatory cytokine and a chemotactic factor that direct polymorphonuclear cells, monocytes, and T cells to inflammatory lesions. IL-31 induces chemokines production and synthesis of IL-6, IL-16, and IL-32. [1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.

Proper citation: Angel Justiz-Vaillant 2020. Copy of ELISA for quantification of IL-30 in human serum.. protocols.io dx.doi.org/10.17504/protocols.io.bj33kqqn Copy   

•   Source: Protocols.io

Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: This kit is designed for the sequential positive selection of CD4+ T cells from human peripheral blood mononuclear cells (PBMCs). Human monocytes express both CD14 and CD4. When using only CD4 Nanobeads for positive selection of human CD4 T cells, monocytes could be isolated along with the T cells. If this monocyte fraction does not impact your application, there is no need to address it. However, not including this population may be required. Thus, the first step in this kit is the depletion of CD14+ cells using a combination of biotin anti-human CD14 and Streptavidin Nanobeads. The second step is the positive selection of the CD4 T cells using directly conjugated CD4 Nanobeads. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water. Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.

Proper citation: Sam Li 2019. MojoSort™ Human CD4 T Cell Selection Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7z8hp9w Copy   

•   Source: Protocols.io

Authors: James Collins, James R. Collins, Krista Longnecker, Helen F. Fredricks, Benjamin A. S. Van Mooy
Group: Van Mooy Lab @ WHOI
Summary: The protocols in this collection were original created by Krista Longnecker and Jamie Collins for creating lipsomes to be used in lipid photo-oxidation experiments. The results of these experiments are detailed in:Collins, J. R, H. F. Fredricks, J. S. Bowman, C. P. Ward, C. Moreno, K. Longnecker, A. Marchetti, C. M. Hansel, H. W. Ducklow, and B. A. S. Van Mooy. 2018. The molecular products and biogeochemical significance of lipid photooxidation in West Antarctic surface waters. Geochimica et Cosmochimica Acta 232:244–264; doi:10.1016/j.gca.2018.04.030and in Chapter 4 of:Collins, J. R. 2017. The remineralization of marine organic matter by diverse biological and abiotic processes. Ph.D. thesis. Cambridge, Massachusetts: Massachusetts Institute of Technology, 300 pp; doi:10.1575/1912/8721

Proper citation: James Collins, James R. Collins, Krista Longnecker, Helen F. Fredricks, Benjamin A. S. Van Mooy 2019. Two-step protocol: Preparation and extrusion of phospholipid liposomes. protocols.io dx.doi.org/10.17504/protocols.io.zbef2je Copy   

•   Source: Protocols.io

Authors: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao
Group: GigaScience Press, BGI, GIGA, GigaScience Press
Summary:  BGISEQ-500 is a new desktop sequencer developed by BGI. Using DNA nanoball and combinational probe anchor synthesis developed from Complete Genomics™ sequencing technologies, it generates short reads at a large scale. 

Proper citation: Jie Huang, Xinming Liang, Yuankai Xuan, Chunyu Geng, Yuxiang Li, Haorong Lu, Shoufang Qu, Xianglin Mei, Hongbo Chen, Ting Yu, Nan Sun, Junhua Rao, Jiahao Wang, Wenwei Zhang, Ying Chen, Sha Liao, Hui Jiang, Xin Liu, Zhaopeng Yang, Feng Mu, Shangxian Gao 2018. BGISEQ-500 Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.pq7dmzn Copy   

•   Source: Protocols.io

Authors: yliu
Group: OnsiteGene 1, XPRIZE Rapid Covid Testing
Summary: This OnsiteGene protocol is designed for testing the normal saliva samples with nucleic acid extraction. A normal saliva collection tube is used for the collection. The protocol uses the Star Array® Hi-SenseTM COVID-19 Molecular Testing Kit 1.0 in the one-step real-time RT-qPCR test assay to qualitatively detect RNA from SARS-CoV-2 virus in the human respiratory specimen. It combines the reverse transcription technology and real-time PCR method to provide accurate detection of the SARS-CoV-2 coronavirus. The Star Array® magnetic beads RNA extraction instrument and kit can automatically extract and purify the RNA for 32 samples in 6 minutes. It is ideal for the saliva samples that contain high amount of PCR inhibitors and can't be processed by the direct amplification. The triplex fluorescence design of the kit simultaneously detects the N1 and Orf1ab genes of the virus and the human RNase P gene as an internal control to ensure the sample quality. This protocol uses the Star Array® XDiveTM Superfast Real-Time PCR instrument to perform 40-cycle PCR in 8 minutes, and can test up to 16 samples or controls in each run. The total protocol time from sample collection to data interpretation is less than 15 minutes.The sample collection steps is adapted from the published protocol:https://www.protocols.io/view/saliva-collection-and-rna-extraction-for-sars-cov-bh6mj9c6

Proper citation: yliu 2020. OnsiteGene 4 Protocol Saliva Extract. protocols.io dx.doi.org/10.17504/protocols.io.bpjemkje Copy   

•   Source: Protocols.io

Authors: Victoria Linderberg
Group: Thomas Crouzier Lab
Summary: This protocol describes how to wash QCM-D sensors before usage and how to modify their surface using a composition of symmetric (oligo)ethylene glycol (OEG) disulfides consisting of 99% dS-OEG and 1% dS-OEG-biotin.

Proper citation: Victoria Linderberg 2016. Protocol for washing and modifying QCM-D sensors. protocols.io dx.doi.org/10.17504/protocols.io.efybbpw Copy   

•   Source: Protocols.io

Authors: David Eccles
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol

Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io dx.doi.org/10.17504/protocols.io.znhf5b6 Copy   

•   Source: Protocols.io

Authors: Mark Dewitt & Julia Wong
Group: Innovative Genomics Institute, CornLab
Summary: This protocol, based on published work, demonstrates how to delivery Cas9 RNP-based gene editing reagents to cultured mamallian cells by electroporation with a Lonza 4d Nucleofector. Consider consulting some of the following papers:1. RNP delivery paper upon which this work is based (Open Access):https://elifesciences.org/content/3/e047662. Paper by an IGI post-doc that details the rationale behind HDR donor design:https://www.ncbi.nlm.nih.gov/pubmed/26789497

Proper citation: Mark Dewitt & Julia Wong 2017. Cas9 RNP nucleofection for cell lines using Lonza 4D Nucleofector. protocols.io dx.doi.org/10.17504/protocols.io.hd8b29w Copy   

•   Source: Protocols.io

Authors: Sonia Hall

Proper citation: Sonia Hall 2016. Blocking Solution (Donkey). protocols.io dx.doi.org/10.17504/protocols.io.fevbje6 Copy   

•   Source: Protocols.io

Authors: Masahiro Ichikawa, Tomoaki Akiyama, Yasushi Tsujimoto, Keisuke Anan, Tadashi Yamakawa, Yasuo Terauchi
Summary: 1.IntroductionMany patients with diabetic mellitus who use insulin develop changes in the subcutaneous adipose tissue at the injection site. These lesions are called lipohypertrophy (LH). The prevalence of LH was reported to be 30-50% or more (1). In a randomized crossover study, injections into sites of LH showed blunted insulin absorption and increased variability compared to injections into normal adipose tissues (2).Since sites of LH reduce the absorption of insulin, patients with LH often use higher total daily doses (TDD) of insulin and have worsened glucose control and higher hyperglycemic hemoglobin (HbA1c) levels (3-5). LH may increase the risk of adverse clinical outcomes and the cost of healthcare.Recently, the effect of injection technique (IT) education to avoid the injection into LH and improve glycemic control on patients with LH has been reported (1, 6).IT education includes stopping injections into LH, stopping needle reuse, and rotating injection sites. However, no meta-analysis has been performed. Thus, to assess the impact of IT education on clinical and metabolic parameters in adults with LH, we will summarize current evidence.2.Research questionP: Patients with type 1 diabetes or type 2 diabetes who are injecting insulin and have LH.I: Injection technique educationC: standard careO: change in TDD3.Method3.1 ProtocolWe used a systematic review protocol template(dx.doi.org/10.17504/protocols.io.biqrkdv6). We followed the Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 for preparing this protocol(7). We will publish this protocol in protocols.io (https://www.protocols.io/).3.2 Inclusion criteria of the articles for the review3.2.1 Type of studiesWe will include individual, cluster, and cross-over randomized trials that assess the injection technique education in patients with type 1 or type 2 diabetes who are injecting insulin and have LH. We will not apply language or country restrictions. We will include all papers including published, unpublished articles, abstract of conference and letter.We will exclude observational studies and non randomized controlled trials. We will not exclude studies based on the observation period or publication year.3.2.2 Study participantsWe will includepatients with type 1 or type 2 diabetes who have been on insulin injections for at least 1 year, with LH, 18 years of age or older, any gender, no HbA1c restriction, and no education about LH within the past 6 months. The diagnosis of LH were made by a physician or nurse when it is clinically visible, palpable, or visible on ultrasound (8). We will accept any number of insulin injections per day, or size of needle or length for insulin injections..We will exclude pregnant women, patients who wish to become pregnant, lactating women and patients taking medications that may cause LH (anti-retroviral or corticosteroid therapy).3.2.3 InterventionIT education: Stop injecting into the LH site, stop reusing needles, and rotate injection sites. We will also accept other definitions of IT education. Any education period is acceptable.3.2.4 ControlStandard care (no intervention, usual care).3.3 Type of outcomes3.3.1 Primary outcomes1.Change in total daily doses of insulinDefinition: Change in total daily doses of insulin from baselinePeriod: Time frame is the longest follow-up period after 3 months2. Change in HbA1cDefinition: Change in HbA1c from baselinePeriod: Time frame is the longest follow-up period after 3 months3. HypoglycemiaDefinition: Hypoglycaemia was defined as the occurrence of one or more symptoms of hypoglycaemia (such as palpitations, tiredness, sweating, strong hunger, dizziness and tremor) and a confirmed blood glucose meter reading of ≤ 60 mg/dL (4). We will also accept hypoglycemia as the original investigators defined.Period: during follow up period3.3.2 Secondary outcomes1. Change in proportion of patients with cured LHDefinition: as original investigators definedPeriod: Time frame is the longest follow-up period after 3 months2. All adverse eventsDefinition: definition of adverse events are set by original authors.Period: during follow up period3.4 Search method3.4.1 Electronic searchWe will search the following databases:1. the Cochrane Central Register of Controlled Trials (CENTRAL);2. MEDLINE via PubMed;3. EMBASE via ProQuest Dialog;See Appendix 1, 2, and 3 for the search strategies.3.4.2 Other resourcesWe will also search the following databases for ongoing or unpublished trials:1. the World Health Organization International Clinical Trials Platform Search Portal (ICTRP);2. ClinicalTrials.gov;See Appendix 4, 5 for the search strategies.We will check the reference lists of studies, including international guidelines as well as the reference lists of eligible studies and articles citing eligible studies. We will ask the authors of original studies for unpublished or additional data.3.5 Data collection and analysis3.5.1 Selection of the studiesTwo independent reviewers will screen titles and abstracts, followed by the assessment of the eligibility based on the full texts. We will contact original authors if relevant data is missing. Disagreements between the two reviewers will be resolved by discussion, and if this fails, a third reviewer will act as an arbiter.3.5.2 Data extraction and managementTwo reviewers will perform independent data extraction of the included studies using standardized data collection form. The form will include the information on the first author’s name, year of study publication, country, sample size, proportion of male participants, mean age of participants, the number of participants with type 1 diabetes mellitus/type 2 diabetes mellitus, estimated diabetic duration, mean duration of insulin treatment, intervention details, detection methods of LH and the outcome measures above. Any disagreements will be resolved by discussion, and if this fails, a third reviewer will act as an arbiter. 3.6 Assessment of risk of bias in included studiesTwo reviewers will evaluate the risk of bias independently using the Risk of Bias 2 tool(9). The effect of interest is the intention-to-treat effect; the effect of assignment to the interventions at baseline, regardless of whether the interventions are received as intended. Disagreements between the two reviewers will be discussed, and if this fails, a third reviewer will be acting as an arbiter, if necessary.3.7 Measures of treatment effectsWe will pool the relative risk ratios and the 95% confidence intervals (CIs) for the following binary variables: Hypoglycemia, proportion of patients with cured LH and all adverse eventsWe will pool the mean differences and the 95% CIs for the following continuous variables: HbA1c, total daily doses of insulinWe will summarize adverse events based on the definition by the original article, but we will not perform meta-analysis..3.8 Unit of analysis issuesClustering at the level of the enrolled units in cluster randomised studies In dealing with cluster-RCTs, for dichotomous data, we will apply the design effect and calculate effective sample size and number of events using the intracluster correlation coefficient (ICC) among each unit and the average cluster size, as described in Chapter 16.3.5 of the Cochrane Handbook (10). If the ICC has not been reported, we will use the ICC of a similar study as a substitute. For continuous data, only the sample size will be reduced; means and standard deviation will remain unchanged (10).Randomised cross-over studiesWe will consider only data from the first period.Multiple comparisonsAll intervention groups that are relevant to this review will be included.3.9 Handling of missing dataWe will ask not-presented data to the original authors.3.9.1 Missing outcomesWe will perform the intention-to-treat (ITT) analysis for all dichotomous data as much as possible.For continuous data, we will not impute missing data based on the recommendation by Cochrane handbook (10). We will perform meta-analysis about the available data in the original study.3.9.2 Missing statisticsWhen original studies only report standard error or p-value, we will calculate the standard deviation based on the method by Altman (11). If we could not obtain these values by contacting authors, standard deviation will be calculated by confidence interval and t-value based on the method by Cochrane handbook (10), or validated method (11). Validity of these methods will be analyzed by sensitivity analysis.3.10 Assessment of heterogeneityWe will evaluate the statistical heterogeneity by visual inspection of the forest plots and calculating the I2statistic (I2 values of 0% to 40%: might not be important; 30% to 60%: may represent moderate heterogeneity; 50% to 90%: may represent substantial heterogeneity; 75% to 100%: considerable heterogeneity). When there is substantial heterogeneity (I2> 50%), we will assess the reason of the heterogeneity. Cochrane Chi2 test (Q-test) will be performed for I2 statistic, and P value less than 0.10 will be defined as statistically significant.3.11 Assessment of reporting biasWe will search the clinical trial registry system (ClinicalTrials.gov and ICTRP) and will perform extensive literature search for unpublished trials. We will assess the potential publication bias by visual inspection of the funnel plot. Egger’s test will be performed as well. We will not conduct the test when we find less than 10 trials or trials which have similar sample size. We will assess the potential publication bias by visual inspection of the funnel plot3.12 Meta-analysisMeta-analysis will be performed using Review Manager software (RevMan 5.4). We will use a random-effects model.3.13 Subgroup analysisTo elucidate the influence of effect modifiers on results, we will evaluate the subgroup analyses of the primary outcomes on the following factors when sufficient data are available.1. age (2. baseline HbA1c 3. gender4. IT education protocols with versus without initial insulin reduction3.14 Sensitivity analysisWe will undertake the following sensitivity analyses for the primary outcomes to assess whether the results of the review are robust to the decisions made during the review process. 1. Exclusion of studies using imputed statistics.2. Exclusion of studies with high overall risk of bias4. Summary of findings tableSummary of findings table will be made for the following outcome based on the Cochrane handbook (10).We will include grading to evaluate the quality of evidence based on the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach for each Summary of findings table (13).1. Change in total daily doses of insulin2. Change in HbA1c3. Hypoglycemia5. Conflict of InterestThe authors declare no conflicts of interests.Appendix 1: CENTRAL search strategy#1[mh Amyloidosis]#2 lipohypertrophy:ti,ab#3 "insulin-derived amyloidosis":ti,ab #4lipos:ti,ab#5 "subcutaneous induration":ti,ab#6"subcutaneous nodules":ti,ab#7 #1 OR #2 OR #3 OR #4 OR #5 OR #6#8 [mh "Injections, Subcutaneous"]#9 [mh "Patient Education as Topic"]#10injection*:ti,ab#11 education*:ti,ab#12 (reus*:ti,ab AND needle*:ti,ab)#13 #8 OR #9 OR #10 OR #11 OR #12#14 #7 AND #13Appendix 2: MEDLINE (via PubMed) search strategy#1 "Amyloidosis"[Mesh]#2 "lipohypertrophy"[tiab]#3 "insulin-derived amyloidosis"[tiab]#4 "lipos"[tiab]#5 "subcutaneous induration"[tiab]#6 "subcutaneous nodules"[tiab]#7 #1 OR #2 OR #3 OR #4 OR #5 OR #6#8 "Injections, Subcutaneous"[Mesh]#9 "Patient Education as Topic"[Mesh]#10 injection*[tiab] #11 education*[tiab] #12 reus*[tiab] AND needle*[tiab]#13 #8 OR #9 OR #10 OR #11 OR #12#14 #7 AND #13#15 randomized controlled trial[pt]#16 controlled clinical trial[pt]#17 randomized[tiab]#18 placebo[tiab]#19 drug therapy[sh]#20 randomly[tiab]#21 trial[tiab]#22 groups[tiab]#23 #15 OR #16 OR #17 OR #18 OR #19 OR #20 OR #21 OR #22#24 animals[mh] NOT humans[mh]#25 #23 NOT #24#26 #14 AND #25Appendix 3: EMBASE (via ProQuest Dialog) search strategy#1 emb(amyloidosis) OR ti(lipohypertrophy) OR ab(lipohypertrophy) OR ti(insulin-derived amyloidosis) OR ab(insulin-derived amyloidosis) OR ti(lipos) OR ab(lipos) OR ti(subcutaneous induration) OR ab(subcutaneous induration) OR ti(subcutaneous nodules) OR ab(subcutaneous nodules) #2 exact(subcutaneous drug administration) OR exact(patient education) OR ti(injection*) OR ab(injection*) OR ti(education*) OR ab(education*) OR (ti(reus*) OR ab(reus*)) AND (ti(needle*) OR ab(needle*)) #3 (ab(random*) OR ti(random*)) OR (ab(clinical NEAR/1 trial*) OR ti(clinical NEAR/1 trial*)) OR (EMB.EXACT("health care quality"))#4 #1 AND #2 AND #3Appendix 4: ICTRP search strategyParticipant: lipohypertrophy OR insulin-derived amyloidosis OR subcutaneous induration OR subcutaneous nodulesIntervention: injection OR education OR (reus AND needle)Appendix 5: ClinicalTrials.gov search strategyCondition or disease: lipohypertrophy OR insulin-derived amyloidosis OR subcutaneous induration OR subcutaneous nodulesIntervention: injection OR education OR (reus AND needle)References1.Chen L, Xing Q, Li J, Zhou J, Yuan Y, Wan Y, et al. Injection Technique Education in Patients with Diabetes Injecting Insulin into Areas of Lipohypertrophy: A Randomized Controlled Trial. Diabetes Ther. 2021.2.Famulla S, Hövelmann U, Fischer A, Coester H-V, Hermanski L, Kaltheuner M, et al. Insulin Injection Into Lipohypertrophic Tissue: Blunted and More Variable Insulin Absorption and Action and Impaired Postprandial Glucose Control. Diabetes Care. 2016;39(9):1486-92.3.Frid AH, Hirsch LJ, Menchior AR, Morel DR, Strauss KW. Worldwide Injection Technique Questionnaire Study: Injecting Complications and the Role of the Professional. Mayo Clinic proceedings. 2016;91(9):1224-30.4.Blanco M, Hernández MT, Strauss KW, Amaya M. Prevalence and risk factors of lipohypertrophy in insulin-injecting patients with diabetes. Diabetes Metab. 2013;39(5):445-53.5.Ji L, Sun Z, Li Q, Qin G, Wei Z, Liu J, et al. Lipohypertrophy in China: Prevalence, Risk Factors, Insulin Consumption, and Clinical Impact. Diabetes Technol Ther. 2017;19(1):61-7.6.Campinos C, Le Floch JP, Petit C, Penfornis A, Winiszewski P, Bordier L, et al. An Effective Intervention for Diabetic Lipohypertrophy: Results of a Randomized, Controlled, Prospective Multicenter Study in France. Diabetes Technol Ther. 2017;19(11):623-32.7. Shamseer L, Moher D, Clarke M, et al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015: elaboration and explanation. BMJ. 2015;349(jan02 1):g7647-g7647. doi:10.1136/bmj.g76478.Deg N,Zhang X, Zhao F, Wang Y, He H. Prevalence of lipohypertrophy in insulin-treated diabetes patients: A systematic review and meta-analysis. 2018;9(3):536-43.9. Sterne JAC, Savović J, Page MJ, et al. RoB 2: a revised tool for assessing risk of bias in randomised trials. BMJ. August 2019:l4898. doi:10.1136/bmj.l489810. Higgins JPT, Thomas J, Chandler J, et al. Cochrane Handbook for Systematic Reviews of Interventions version 6.0 (updated July 2019). Cochrane, 2019.11. 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Proper citation: Masahiro Ichikawa, Tomoaki Akiyama, Yasushi Tsujimoto, Keisuke Anan, Tadashi Yamakawa, Yasuo Terauchi 2021. Efficacy of injection technique education in diabetes with lipohypertrophy: a systematic review and meta-analysis protocol. protocols.io dx.doi.org/10.17504/protocols.io.bteenjbe Copy   

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