The importance of sample quality for qPCR

The simplicity of assay design and execution, together with sensitivity and specificity have made this the method of choice for nucleic acid quantification that is reflected by the prodigious number of peer-reviewed publications reporting qPCR data. Inevitably, a corresponding number of different protocols, reagent recipes, analysis methods and reporting formats are also being used, which is resulting in the scientific literature being corrupted with publications reporting insignificant and conflicting results. The inexperienced or casual qPCR user is vulnerable to the production of inaccurate data because even assays of extremely poor quality usually yield results that are amenable to statistical manipulation.

Technical deficiencies that affect assay performance include:

• Inadequate sample storage, preparation and nucleic acid quality, resulting in highly variable results
• Poor choice of primers for reverse transcription and adoption of inappropriate protocols for quantitative measurements
• Poor choice of primers and probes for PCR
• Inadequate assay optimisation leading to inefficient and unstable assays
• Inappropriate data
• Statistical analyses, generating results that can be highly misleading

In this series of six articles, each of the technical challenges listed will be addressed and recommendations for best practice will be provided.

Sample storage

Tissue sample acquisition constitutes the first, potential source of experimental variability. To date there are almost as many tissue collection and storage protocols as there are pathologists, with little standardisation of procedures.

Yet as these samples are used for ever increasingly sophisticated analysis it is important to verify the variability introduced during sample handling. This is particularly important for experiments targeting RNA. While DNA is remarkably stable, transcript molecules are incredibly unstable and so mRNA profiles may be perturbed during sample collection and processing. Consequently, in order for more accurate interpretation of scientific reports it is important to record in detail the nature of the samples and extraction, where tissue was obtained from, whether it was processed immediately and, if not, how it was preserved and how long and under what conditions it was stored.

As a general guide the faster that nucleic acid, particularly RNA, is stabilised the better, therefore sample preparation and speed of extraction are key to successful RNA extraction. Conventionally RNA extracted from tissue or cells requires that samples are processed immediately after collection from the physiological source (fresh tissue) or are snap frozen in liquid nitrogen (fresh frozen). However there are practical issues surrounding either of these approaches: The first consideration for the surgeon is the health of the patient and not the demands of the impatient molecular biologist, hence clinical material may be left at room temperature for several minutes or hours prior to stabilisation. Under field conditions continuous refrigeration is rare and so sample storage and transport is a major concern. After freezing the tissue, RNA must be extracted while preventing the sample from defrosting. In an extremely laborious process, most often the tissue is then ground to a fine powder while liquid nitrogen is continuously added. Many companies are developing products to address these challenges. The first of these is RNAlaterTM (12, available from various vendors including Sigma Aldrich, Qiagen, Ambion), which has been closely followed with related solutions to address specific sample types. This is a high salt concentration solution at pH5.2 including 70% ammonium sulphate. High concentrations of ammonium sulphate diffuse into tissue samples that are placed into the storage solution. This results in precipitation of cellular proteins thus protecting cellular RNA from RNAse activity after cell lysis. Numerous studies have compared RNA integrity after permutations of storage in liquid nitrogen, frozen at -80°C, -20°C, 4°C, room temperature and each of these temperatures after immersion into the RNAlaterTM storage solution also taking note of the effect of surface area and diffusion on effective RNAlaterTM preservation. In most cases RNA is preserved when samples are stored in liquid nitrogen, at -80°C or in RNAlaterTM and in one study it was shown that more data variability was due to patient variability than to sample handling13. However, there are also reports of loss of transcript proportionality after sample storage in RNAlaterTM and some suggestion that fresh tissue can be stored on ice without major effects on RNA quality and expression14 demonstrating that it is still critical to validate the sample acquisition and storage protocol for each experimental set up.

Nucleic acid preparation

After collecting the required samples the next critical step is to extract desired nucleic acid (and possible proteins as well). Extraction efficiency, and therefore nucleic acid yield, depends on factors relating to the sample e.g. cell culture or solid tissue, physiological status (e.g. normal, cancerous or necrotic), genetic complexity as well as technical considerations such as adequate sample homogenisation, the total mass processed and the relative nucleic acid to cells ratio and the presence of RNases in the tissue. In order to extract high quality nucleic acid it is critical to minimise the activity of nucleases upon cell lysis. In many cases tissue disruption is accompanied by simultaneous addition of a strong protein denaturation agent containing a chaotropic compound such as guanidinium salt. This serves to inactivate nucleases and solubilise cell membranes. Lysis buffer can be added directly to adherent cells after removal of growth media, avoiding trypsinisation whenever possible. When isolating RNA in particular, it is critical that frozen tissue samples do not defrost during the process of cell disruption. Particularly challenging sample types include formalin fixed and paraffin fixed archival sections. These are typically treated with proteinase K to degrade the tissue structure prior to guanadinium based extractions. The nucleic acid purified from these samples is usually very fragmented, around 200 bases, and RNA is challenging to work with due to crosslinking between bases and also to proteins15,16. Clearly the most appropriate extraction process should also be validated for each sample type.

Nucleic acid quality control

In order to achieve qPCR or RT-qPCR data that are robust, reliable and ultimately reveal useful scientific information it is essential to analyse the highest quality nucleic acid possible. The major factors to consider are concentration, purity and integrity.

While some considerations apply equally to both RNA and DNA e.g. purity there are some factors that have greater applicability to one template.

RNA samples

The quality of RNA should be determined as a matter of routine whenever possible. This may not be applicable when quantity of total RNA extracted is too low to permit quality assessment such as when working with single cells, or where extraction and RT-qPCR steps are performed as a continuous, one-tube experiment.

Since it is advisable to use approximately the same amount of RNA for cDNA synthesis when comparing different samples, quantification of RNA in extracted samples is important. There are several procedures for the measure of nucleic acid concentration including spectrophotometric or Nanodrop analysis, the Agilent BioAnalyser/BioRad Experion/Qiagen QIAexcel, or Ribogreen fluorescence detection. Unfortunately each system produces a different result and so it is imperative that a single system is selected and data obtained using the different methods are not directly compared17 especially when performing reverse transcription reactions, when it is usually important to include the same concentration of RNA. The purity of the sample includes a consideration of the extent of genomic DNA (gDNA) contamination that can interfere with RT reactions and also cause an increased estimate of copy number (depending on design and relative concentration of contamination and transcript concentration). It is advisable to treat RNA samples with DNAse, with in solution protocols being more effective but risking loss of material and on column systems reducing losses but less efficient at gDNA removal. The selected DNAse protocol will depend upon sample size and personal preference. It is advisable to establish that the RNA sample does not contain factors that may inhibit downstream RT reactions or PCR. This is particularly important because assays are not inhibited equally and therefore any inhibition will result in loss of transcript proportionality18. Measurement of OD260 /OD280 absorbance ratios must be performed in a pH neutral buffer, but is not sufficient for quantitative analysis, especially when the aim is to measure minor differences (less than 10-fold) in mRNA expression levels. It does provide an indication of RNA purity, since presence of DNA or residual phenol alters the ratio. A more accurate determination can be established by serial dilution of the sample establishing that the appropriate increase in Cq (19; Ct or Cp) is recorded or using a universal inhibition assay such as SPUD20. It is critical to determine the integrity of the RNA because even partial degradation results in reduced sensitivity for detecting low-level transcripts21 and different relative degradation of transcripts will result in determination of incorrect target ratios. While significant RNA degradation takes place in vivo as a normal consequence of the natural regulation of mRNAs in response to environmental stimuli22. This kind of degradation is outside the control of the researcher; one effect of this kind of degradation is that even RNA samples shown to be of high overall quality could show differential degradation for individual mRNAs. Integrity should be analysed at the very least using a gel-based system or microfluidics-based rRNA analysis23 or preferably using reference gene/target gene 3’:5’ integrity assay24. The Bioanalyser/Experion systems calculate RIN or RQI numbers that provide some information about the general state of the RNA sample, although these are not an absolute measure of quality. The 3’:5’ assay requires that the PCR efficiencies of both assays are virtually identical (as shown in reference25) or that the quantities are measured against an external standard curve and that they are not subject to differential inhibition; it also necessitates the establishment of a threshold criterion that delineates RNA of sufficient quality to yield reliable results. Ideally, the assay should target a panel of “integrity reference genes”, probably intron-less, with a 3’:5’ threshold ratio of around 0.2-5.

DNA samples

In general, degradation is much less of an issue with DNA; however it is important to be able to assess the extent of DNA degradation for forensic applications, where biological samples collected from crime scenes, mass disasters, and missing persons cases may have been exposed to harsh environmental conditions that break down the chemical structure of DNA. The small amplicon size of qPCR assays helps to minimise assay-related problems, but methods have been developed that provide a quantitative measurement of DNA quality26 and should be considered for such specialised purposes.

Awareness of potential inhibition is as critical for DNA analysis as it is for RNA. It is important to ensure that no inhibitors have been co-purified with the DNA that will distort, for example, pathogen detection and quantification18. As described for analysis of RNA samples, different PCR reactions are not equally susceptible to inhibition by substances co-purified in nucleic acid extracts27,28 hence detection of inhibitors by inclusion of external spikes 30 but the general use of dilutions of nucleic acids to demonstrate the relative decrease in that Cqs or copy numbers is less vulnerable to assay specific inhibition.

Conclusion

The quality of the qPCR and RT-qPCR assay is dependent on the quality of the sample. Each stage of the sample extraction, storage and preparation contributes to the quality of the nucleic acid and should be considered individually in order to ensure that the highest quality material is analysed.

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