Quantitative PCR

Quality Control: Methods

A protocol should be established to minimize the variability between sample collections and subsequent qPCR analyses. In addition, Bustin et al. (2009) describe guidelines for the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) that are important for data quality assessment. Important factors include:

Sample Collection

1. Sample source
2. DNA or RNA extraction method: kits, modificaitons, reagents, DNase or RNase treatment, contamination assessment
3. If sampling RNA, reverse transcription method: reaction conditions, amount of RNA and reaction volume, reverse transcriptase concentration, temperature and time
4. Transportation, storage and handling methods

qPCR

1. Target gene: symbol, sequence accession number, length of sequence to be amplified (amplicon)
2. Oligonucleotide primers: sequence, specificity
3. Reagents list
4. Reaction conditions: volume, amount of DNA or cDNA, polymerase identity and concentration, buffer kit manufacturer, thermocycling parameters
5. Aliquot standards to minimize thawing and refreezing

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