Additional files
Additional file 1
Sample/Templ Details
ate
Source
If cancer, was biopsy
screened for adjacent
normal tissue?
Method of
Liquid N2; RNAlater;
preservation
formalin
Storage time
If using samples >6 months
(if appropriate) old
Handling
fresh; frozen; formalin
Extraction
TriZol; columns
method
RNA: DNAIntron-spanning primers; no
free
RT control
Concentration
RNA: integrity
Inhibition-free
Assay
optimisation/v
alidation
Accession
number
Amplicon
details
Primer (and if
used probe)
sequence
amplicon
verification
In silico
empirical
Priming
conditions
PCR efficiency
Linear
dynamic range
Limits of
detection
Intra-assay
variation
Nanodrop; Ribogreen;
microfluidics
Microfluidics; 3':5' assay
Method of testing
RefSeq XX_1234567
Checklist
Ostearthritic tissues, conifmred by histology
and radiography
RNAlater
<6 months
Fresh
Trizol/Chloroform and Rneasy columns
Dnase on-column treatment, intron spanning
primers where possible, genomic
contamination RT/PCR check
Nanodrop
RIN (Ref 48)
Not done
Table 1
exon location, amplicon
Table 1
size, amplicon sequence
even if previously published Table 1
melt-curves
BLAST; Primer-BLAST;
m-fold
primer concentration;
annealing temperature
oligo-dT; random;
combination; target-specific
dilution curve
spanning unknown targets
LOD detection; accurate
quantification
copy numbers not Cq
Probes used, Agarose gel confirmation of
amplicon size
BLAST
Concentration standardised, in Material and
Methods section
Oligo dT
>93% <107%
1-1000 dilution
Not done
Not done
RT/PCR
Protocols
Reagents
Duplicate RT
NTC (no
template
controls)
NAC (no
amplification
controls)
Positive
control
Data analysis
Statistical
justification
Transparent,
validated
normalisation
Specialist
software
detailed description,
concentrations, volumes
supplier, Lot number
DCq
Cq & melt curves
Described in Materials and Methods section
DCq beginning:end of
qPCR (only for probe-based
reactions)
inter-run calibrators
Not done
e.g., biological replicates
Duplicate
e.g., GeNorm summary
GeNorm
e.g., QBAsePlus
Not required
Described in Materials and Methods section
Single RT
Included
Not done