Variables Affecting qRT-PCR Success

The two most significant variables that affect the performance of qRT-PCR are the presence of inhibitors and the presence of genomic DNA. Inhibitors can affect enzyme performance and can lead to a decrease in sensitivity. gDNA in the qRT-PCR reaction can cause false positive results and background. Another less -understood variable in qRT-PCR performance is RNA quality.

Monitoring Inhibition During qRT-PCR

Inhibition of qRT-PCR can decrease the sensitivity of detection, resulting in false negative results, but can also result in the inability to accurately quantitate the RNA of interest.

Most often, inhibition is due to the carryover of chemicals or compounds from the sample preparation process. Examples of some common inhibitors are heme, detergents, and salts. Strict adherence to the sample preparation process, including washes, can reduce the chance that inhibitors are carried over.

For some sample preparation reactions, reagents are left in the sample, which do not have an effect at low concentrations but at higher concentrations can cause inhibition. Transfer of too much sample into the qRT-PCR reactions can result in inhibition via this mode.

Monitoring for inhibition is potentially important for every sample that is analyzed and can be done using a variety of tools. One such tool is the Xeno™ RNA control. This is an exogenous RNA that is not similar to any sequence present in available databases. This RNA can be added to the reverse transcription reaction and then detected during qPCR. Identical results for Xeno™ RNA in reactions with and without sample can confirm the lack of inhibitors. An increase in Ct for both Xeno™ RNA and a target confirm that inhibitors are present (Figure 1).

 

Figure 1. (A) A titration of two different cell types prepared using the Cells-to-CT ™ kit protocol. Xeno™ RNA, an exogenous control RNA, is added to the RT reactions to assess monitor for inhibition. qPCR was performed to assay two genes of different expression levels (ACTB and VEGF) and Xeno™ RNA. The linearity of the target mRNA transcript signal and the maintenance of the Ct for Xeno™ RNA indicate that, as cell number increases, no inhibition occurs. (B) In a similar experiment using a different cell line (known to carry qRT-PCR inhibitors), neither the linearity constant value of the Xeno™ RNA signal nor the linearity of the target mRNA signal were maintained, showing that, as cell number increases, inhibition of the qRT-PCR reactions also increases.

Effects of RNA Integrity on qRT-PCR

It is generally thought that high-quality, undegraded RNA is required for all types of analysis. Though this may be true for microarrays or for gel-based protocols such as northern blot analysis, it is not necessarily true for qRT-PCR. Many of the primer/probe sequences used for qRT-PCR are quite short, ranging from ~50 bases to ≥200 bases, allowing even significantly degraded RNA to be used for analysis, without detectable effects. Table 1 shows RIN values from RNA analyzed on the Bioanalyzer™ platform (Agilent Technologies) and correlates them to Ct values obtained from qRT-PCR. It is clear that neither 1-step nor 2-step qRT-PCR reactions are affected until a RIN value of approximately 5 is reached.

Purified RNA average RIN1-step qRT-PCR Ct2-step qRT-PCR Ct
9.825.3821.81
8.525.3221.82
5.125.3021.89
2.533.75

24.76


Table 1. RNA was degraded using a variety of methods and analyzed on an Agilent Bioanalyzer™ instrument to determine RIN values. qRT-PCR was then performed using 1-step and 2-step reactions.

All RNA Isolation Methods Yield RNA Containing Residual Genomic DNA

We have found that genomic DNA must be actively removed via DNase I digestion, acid phenol:chloroform extraction, or lithium chloride precipitation. To illustrate this, qRT-PCR was performed on HeLa RNA isolated by several different methods (Table 2).

Sample
Kit+RT Ct–RT Ct
ΔCt
105 HeLa cellsMagMax™-96 Total RNA Isolation Kit
(without DNase digestion)
26.5131.525.01
105 HeLa cellsMagMax™-96 Total RNA Isolation Kit
(with DNase digestion)
26.6239.6313.01
105 HeLa cellsPureLink® RNA Mini Kit
(without DNase digestion)
26.6827.951.27
104 HeLa cells   TaqMan® Gene Expression Cells-to-CT™ Kit
(without DNase digestion)

30.14

32.882.74
104 HeLa cellsTaqMan® Gene Expression Cells-to-CT™ Kit
(with DNase digestion)   
30.1040.009.90

Table 2. Isolating gDNA-free RNA. RNA from 100,000 or 10,000 HeLa cells was prepared using various kits, and RNA was detected using qRT-PCR. Differences in ΔCt (without vs. with DNase treatment) are evidence of gDNA removal. Samples were tested using the MagMax™ and PureLink® kits with the Assays-on-Demand® products for FOXD1 (Hs00270117_s1), and the Cells-to-CT™ kit with the corresponding products for EEF1A1 (Hs00742749_s1).

DNase I Treatment Is Most Effective

DNase I digestion has consistently proven to be the most effective method for removing DNA contamination from RNA samples. Table 3 and Figure 2 demonstrate how effective DNase I treatment can be on purified RNA samples analyzed by qRT-PCR. Purified RNA samples were analyzed, pre- and post-treatment with the TURBO DNA-free™ kit, using a TaqMan® assay that detects gDNA.

  +RT Ct–RT Ct
ΔCt
Purified RNA, not treated with the TURBO DNA-free™ kit 27.0130.443.43
Purified RNA, treated with the TURBO DNA-free™ kit 26.6840.0012.99

Table 3. gDNA digestion after purification. RNA was treated with the TURBO DNA-free™ kit, and qPCR was performed using the Assays-on-Demand® product for FOXD1 (Hs00270117_s1). Differences in ΔCt (without vs. with DNase treatment) are evidence of gDNA removal.

 

Figure 2. Effect of DNase I digestion on Ct values. Total RNA isolated from HeLa cells was converted to cDNA and assayed using qPCR for FOXD1. The amplification plot shows no detectable signal from –RT samples treated with TURBO DNase™, whereas –RT samples without DNase treatment have detectable signal, due to gDNA.

Using Intron-Spanning Primers and Probes to Avoid gDNA Detection

Genes are divided into coding sequences, called exons, and noncoding sequences, called introns. Intron sequences are removed after a pre-mRNA is transcribed to create a contiguous coding sequence. gDNA detection during PCR can be effectively eliminated by selecting primers and probes that span an intron/exon boundary. When primer and probe sets (used for TaqMan® analysis) or primer sets (used for SYBR® Green I dye analysis) are designed within a single exon, amplification and detection can occur from the mRNA or the gDNA sequences. The Applied Biosystems® TaqMan® assays that are designed in this way have a suffix _s1 (e.g., Hs00328611_s1, Figure 3). Other assays are designed to minimize gDNA detection with their primer and probe sequences located in different exons. This allows amplification of the correctly sized sequence from the mRNA, but depending on the location of the two exons and their separation, amplification from gDNA may also occur. These assays have a suffix _g1 (e.g. Hs01064647_g1, Figure 3). In order to eliminate amplification from gDNA, other assays are designed with their probe complementary to the splice junction. In this way, only the spliced mRNA can be detected during qRT-PCR. These assays have a suffix of _m1 (e.g. Hs01056318_m1, Figure 3).

 

Figure 3. The Alignment Map feature in the TaqMan® Gene Expression Assays Search portal showing the interleukin 17 receptor A gene. Shown are six different annotated transcripts, with their exons in blue. Gene expression assays are shown in red. Highlighted are an _m1 assay, which has its probe spanning an exon junction (so it will not amplify gDNA); a _g1 assay, which has its probe and primers in two different exons (possibly allowing it to amplify gDNA); and an _s1 assay, which has its primers and probe within a single exon (definitely allowing it to amplify gDNA).

Simplifying RNA Isolation for qRT-PCR

One of the key steps that affect the success of any qRT-PCR experiment is RNA isolation. RNA isolation methods such as acid phenol extractions, purification using glass-fiber filters or magnetic beads, and lysis-based reagents, can each provide RNA of acceptable quality for qRT-PCR.

Life Technologies has developed a variety of Ambion® and Invitrogen™ products for efficient gDNA-free RNA isolation. Also offered are reagents that can remove genomic DNA post-purification (Figure 1).

TRIzol® Reagent is a ready-to-use monophasic solution of phenol and guanidine isothiocyanate, suitable for isolating total RNA from cells and tissues. During sample homogenization or lysis, TRIzol® Reagent helps to maintain the integrity of the RNA while disrupting cells and dissolving cell components.

The MagMAX™-96 Total RNA Isolation Kit utilizes MagMAX™ technology to purify total RNA on magnetic beads in a tube or 96-well format. The RNA is eluted and then digested with Ambion® RNase-free TURBO DNase™ I to rid the sample of genomic DNA contamination. The sample is then bound again, cleaned, and eluted—ready for analysis.

The PureLink® RNA Mini Kit uses column-binding technology to purify RNA in a microcentrifuge tube format. After cell lysis, the sample is added to the column and the RNA binds to the membrane. A DNase digestion step is subsequently performed on the membrane. RNA is then eluted purified and gDNA-free.

The Cells-to-CT™ Kits offer a method for preparation from cultured cells of RNA for qRT-PCR that doesn’t involve an RNA purification step. Cell lysis and DNA digestion take place in a single 5-minute reaction at room temperature. The lysis and DNase I digestion are then inactivated with the Stop Solution for 2 minutes at room temperature. After this 7-minute preparation, the RNA is ready for analysis by qRT-PCR.               

For removing gDNA from a previously isolated RNA sample, the TURBO DNA-free™ kit provides an efficient solution. This kit combines the highly active TURBO DNase™ I enzyme and a novel reagent: TURBO DNA-free™ DNase Removal Reagent. This reagent removes the TURBO DNase™ I and divalent cations rapidly and effectively, eliminating the need to heat-inactivate the enzyme, which can lead to strand scission of the RNA. TURBO DNA-free™ DNase Removal Reagent also helps to reduce protein contamination and removes the need to use phenol:chloroform extraction, which can reduce the RNA yield from limiting samples. 

 

Figure 4. Picture of a variety of Trizol, MagMAX, Purelink, C2Ct and TURBO DNA-free

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