Method of processing and preservation: How was the sample treated and stored?
... Step 2: Reverse Transcription. For optimal results, use: Roche Transcriptor ...
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Experiment title: Performed by: Date: Institution: Step 1: Sample Preparation & Nucleic Acid Isolation For great results, use (click product names to learn more): Roche High Pure RNA Isolation Kit Roche High Pure FFPET RNA Isolation Kit Roche High Pure miRNA Isolation Kit Roche RealTime ready Cell Lysis Kit
From which source (animal, organ, tissue) does the examined material originally come from? Which volume or mass or cell number was used for nucleic acid preparation?
Experimental design: How did you choose and set up your study (number of treated samples and controls)
Handling: Which tools or methods were used to obtain and process the primary samples (e.g., microdissection, macrodissection)?
Method of processing and preservation: How was the sample treated and stored?
If frozen – how and how quickly?
If fixed – with what, and how quickly?
If stored for longer: how and how long? (especially for FFPE samples)
Extraction method: Which kit or instrument was used to extract/isolate the DNA/RNA from the starting material? Roche High Pure RNA Isolation Kit, High Pure FFPET RNA Isolation Kit, High Pure miRNA Isolation Kit, RealTime ready Cell Lysis Kit, or other (Please specify)
Was the vendor’s protocol modified (If Yes, when, and how? e.g. by using additives)
Did you do a DNAse or RNAse treatment? (If Yes, when?)
Did you check for nucleic acid purity and integrity? If Yes: By using which instrument and method? What was the resulting purity (A260/A280)? What was the resulting yield? If No: Why not?
Did you check for the presence of PCR inhibitors? If Yes: By using what (e.g. Cq dilutions, spike or other (please specify)If No: Why not?
Final storage solution (e.g., buffer, H2O) for the purified total RNA:
Storage time and temperature of the purified total RNA before use in RT-qPCR:
Step 2: Reverse Transcription For optimal results, use: Roche Transcriptor First Strand cDNA Synthesis Kit Roche Transcriptor Universal cDNA Master
Amount of RNA and reaction volume:
Priming oligonucleotide (if using gene specific primers) and concentration:
Reaction temperature and time:
Manufacturer of reverse transcription reagent(s) and catalogue number(s):
Reverse transcriptase type and used concentration:
Storage conditions of cDNA:
Step 3: PCR Amplification and Analysis For best results, use: LightCycler® 480 Probes Master FastStart Essential DNA Probes Master FastStart Universal Probe Master (Rox)
Target sequence and amplicon information: Target gene database sequence accession number:
Location of amplicon:
Amplicon length:
Result of in silico specificity screen (BLAST, etc.):
Information on pseudogenes, retropseudogenes or other homologs:
Secondary structure analysis of amplicon:
Determined by which method?
Location of each primer relative to exons or introns (if applicable):
Targeted splice variants:
Primer sequences:
RTPrimerDB Identification Numbers:
Manufacturer of oligonucleotides:
Purification method:
For probe-based assays: Probe type:
Probe sequence(s):
qPCR reaction conditions Reaction volume and amount of cDNA/DNA per reaction:
Primer, (probe), Mg2+ and dNTP concentrations:
Polymerase identity:
Buffer/kit manufacturer and identity (e.g., catalog number)
Manufacturer and catalog number of plates or tubes and catalog number:
Complete thermocycling parameters:
Reaction setup: Was it manual or robotic? If robotic: Using which robot?
Equipment: Which Real-Time PCR instrument was used? (Which Roche LightCycler® System or other (please specify)?)
Validation of qPCR runs: Are you running a multiplex assay? If yes, please describe efficiency and limit of detection for each assay:
How did you check for specificity of amplification for each target (e.g., on a gel, by sequencing, melting curve analysis or digest):
For SYBR Green I assays: Cq of the non-template control reaction:
Standard curve characteristics (slope and y-intercept):
How many replicates did you use to establish the standard curve?(xx replicates per standard concentration)
What was the lower and the upper limit of the standard curve?
PCR efficiency calculated from slope:
Confidence interval for PCR efficiency or standard error:
r2 of standard curve:
Information on linear dynamic range:Cq variation at lower limit:
Confidence intervals throughout range:
Evidence for limit of detection:
How many reactions per run were used for controls? (please specify positive and negative controls, controls without template and No RT controls, e.g. Positive controls: 3 reactions in 5 replicates per 96 well plate)
Data analysis: Vendor software: Which software type, version and algorithm provided by the PCR machine supplier was used to analyze the data?
Specialist software: Which (if any) additional software was used? Self-developed algorithms, or other (please specify)
Normalisation: Which reference gene(s) were used to calculate the relative expression of the studied genes?
What was the reason for choosing these particular genes?
Which algorithm (e.g., geNorm, bestkeeper, normfinder) was used to normalize for reference gene(s)
Which principle was used for Cq calling?
What was the number and of biological replicates used?
How was their concordance?
How many technical replicates were used, and at which step (RT or qPCR)?
What was the observed repeatability (intra-assay variation)?
What was the observed reproducibility (inter-assay variation, %CV)
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