|
Source
|
If cancer, was biopsy screened for adjacent normal tissue?
|
|
Method of preservation
|
Liquid N2/RNAlater/formalin
|
|
Storage time (if appropriate)
|
If using samples >6 months old
|
|
Handling
|
fresh/frozen/formalin
|
|
Extraction method
|
TriZol/columns
|
|
RNA: DNA-free
|
Intron-spanning primers/no RT control
|
|
Concentration
|
Nanodrop/ribogreen/microfluidics
|
|
RNA: integrity
|
Microfluidics/3':5' assay
|
|
Inhibition-free
|
Method of testing
|
|
Assay optimisation/validation
| |
|
Accession number
|
RefSeq XX_1234567
|
|
Amplicon details
|
exon location, amplicon size
|
|
Primer sequence
|
even if previously published
|
|
Probe sequence*
|
identify LNA or other substitutions
|
|
In silico
|
BLAST/Primer-BLAST/m-fold
|
|
empirical
|
primer concentration/annealing temperature
|
|
Priming conditions
|
oligo-dT/random/combination/target-specific
|
|
PCR efficiency
|
dilution curve
|
|
Linear dynamic range
|
spanning unknown targets
|
|
Limits of detection
|
LOD detection/accurate quantification
|
|
Intra-assay variation
|
copy numbers not Cq
|
|
RT/PCR
| |
|
Protocols
|
detailed description, concentrations, volumes
|
|
Reagents
|
supplier, Lot number
|
|
Duplicate RT
|
ΔCq
|
|
NTC
|
Cq & melt curves
|
|
NAC
|
ΔCq beginning:end of qPCR
|
|
Positive control
|
inter-run calibrators
|
|
Data analysis
| |
|
Specialist software
|
e.g., QBAsePlus
|
|
Statistical justification
|
e.g., biological replicates
|
|
Transparent, validated normalisation
|
e.g., GeNorm summary
|
