Biomedical Engineering Reference
In-Depth Information
Sample selection
(fresh, frozen, archival)
Pre-
Tissue selection
(whole biopsy, microdissection)
assay
RNA preparation
(extraction, no extraction)
RNA quality assessment
(integrity, inhibitors, concentration)
Reverse transcription
(priming method, RTase)
Assay
PCR chemistry
(probe, non-probe)
Data analysis
(thresholds, normalisation, interpretation)
Figure 6.1
RT-qPCR experimental workflow. The assay divides into two main segments, the
pre-assay and the assay, which can be subdivided into sample selection, tissue selection, RNA
preparation, RNA quality assessment and reverse transcription (RT), PCR chemistry, data analysis
respectively. These are highlighted in different colours.
monitoring and quantification of gene expression changes [45]. However, it is essential to
realize that quantification from very few cells also poses problems, as different cells of the
same type may not express the same set of mRNAs [46]. In addition, it could be expected
that identical cell samples would contain identical copy numbers of a target mRNA, or more
likely a normal distribution; however, it has been demonstrated that mRNA distribution is
often in a lognormal fashion [47]. Microdissection can be carried out on either fresh/frozen
or archival samples.
6.2.2 RNA extraction
RNA extraction is easiest from fresh or frozen material, with the main concern relating
to the maintenance of RNA integrity. RNA is easily degraded and it is easy to co-purify
inhibitors of the RT, or PCR steps, which will generate inaccurate results [48]. When deal-
ing with very small amounts of sample (e.g. single cells or minute amounts of laser capture
microdissected fresh/frozen sample) it is best to carry out the RT-qPCR directly from lysed
tissue without going through an extraction process [49]. In our experience this results in an
RNA quality indistinguishable from that obtained using conventional purification methods
(see Figure 6.2). RNA integrity is best determined using a 3
:5
assay, either target specific
or using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the target sequence [11]
