High-quality RNA for RT-PCR using the Absolutely RNA RT-PCR miniprep kit
Karen Dolter Jeff Braman
Stratagene has determined that standard RNA purification kits are not satisfactory for reverse transcribed PCR (RT-PCR) applications. We tested several commercially available RNA purification kits and have shown that contaminating DNA will produce false positive amplicons during RT-PCR. The Absolutely RNA RT-PCR miniprep kit eliminates contaminating DNA and produces highly pure total RNA qualified for RT-PCR reactions from 5 to 40 mg of tissue and 10 5 to 10 7 cultured cells. The Absolutely RNA RT-PCR miniprep kit utilizes a solid-phase DNase treatment to reduce DNA contamination to levels undetectable by PCR.
RT-PCR is an important technique for gene discovery and gene expression analysis because it amplifies expressed RNA sequences.1 RT-PCR is very sensitive; it facilitates the investigation of rare mRNA transcripts that are difficult to detect by other means in studies in which the quantity of starting material is limited. High yield of RNA is important because diminished or biased recovery may result in the loss of rare mRNA. Extreme purity is critical because contaminating DNA present in the RNA sample can give rise to amplification products that mimic the amplification product expected from the RNA target. The Absolutely RNA RT-PCR miniprep kit eliminates these problems by providing high yields of total RNA with undetectable levels of DNA from animal tissues and cultured cells.2
In Figure 1, PCR analysis was performed to demonstrate the absence of DNA contamination in RNA isolated from different quantities of mouse tissue and cells. Using the Absolutely RNA kit, total RNA was isolated from mouse liver and mouse NIH/3T3 cells. From 5, 20, and 40 mg of liver, RNA yields were 14, 37, and 100 g, respectively, and from 105, 106, and 107 cells, RNA yields were 2, 19, and 142 g, respectively. Using Stratagenes prostar first strand RT-PCR kit3, cDNA was synthesized, and control reactions were run in the absence of reverse transcriptase. A 1-l aliquot of each of these reactions was used in PCR (Methods). The PCR included primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) related sequences that are present in greater than 200 copies in the mouse and rat genomes.4 The generation of a 0.2-kb PCR product from the control samples lacking reverse transcriptase (RT) indicates that DNA is present in the RNA sample. The absence of PCR product in the RT minus samples demonstrates that DNA is not detectable in the RNA purified with the Absolutely RNA kit.
Total RNA was isolated from tissue and cultured cells using several commercial kits, then examined by RT-PCR to determine which samples contained DNA contamination. RNA was isolated using Ambions RNAqueous kit, Clontechs NucleoSpin RNA kit, Qiagens RNeasy kit and Stratagenes Absolutely RNA RT-PCR miniprep kit, according to the instructions provided for each. An additional kit (Boehringer Mannheims High Pure RNA Isolation kit) was omitted from this comparison because it is not suited for tissue samples and is limited to RNA isolation from no more than 106 cells.
Stratagenes ProSTAR first strand RT-PCR kit was used to synthesize cDNA3 and control reactions were run in the absence of reverse transcriptase. An aliquot of each of these reactions was used in PCR (Methods). RNA isolated using the kits from Ambion, Clontech, and Qiagen contained DNA contamination, as shown by the presence of a 0.2-kb PCR band in the control lanes (Figure 2). In contrast, the RNA isolated using the Absolutely RNA kit shows no evidence of DNA. This result demonstrates that the Absolutely RNA miniprep kit is effective at eliminating DNA contamination and competitive kits are not.
Recovery of RNA from all abundance classes is important for accurate assessment of gene transcription levels. In addition, preservation of the distribution and abundance of mRNAs is important for generating optimal cDNA libraries. RT-PCR was used to assess the presence of low-, medium-, and high-abundance classes of mRNA (corresponding to approximately 0.1% or less, 0.1 to 1%, and 1% of total RNA, respectively)5 in total RNA isolated from mouse liver and testis using the Absolutely RNA kit.
For the experiment shown in Figure 3, the ProSTAR first strand RT-PCR kit was used to synthesize cDNA from the RNA.3 The resulting cDNA was used in PCR with Stratagenes control RT-PCR primers for genes of varying abundance levels in the mouse.5,6 These include primers for high-abundance transcripts (mouse elongation factor 1a), medium-abundance transcripts (b2-microglobulin), and low-abundance transcripts (protein phosphatase 1 and ornithine decarboxylase). Although b2-microglobulin mRNA is classified as medium abundance, its expression level is higher in liver than in brain7 (Figure 3). Similarly, ornithine decarboxylase is expressed at overall low levels but the levels vary in tissues throughout the body.8 The presence of the expected PCR bands in Figure 3 shows that even rare RNA transcripts are represented in total RNA isolated with the Absolutely RNA kit.
RNA isolated with Stratagenes Absolutely RNA RT-PCR miniprep kit is ideal for RT-PCR. The kits quick, convenient protocol includes a DNA removal step that ensures sensitive RT-PCR results without having amplification products generated from genomic DNA targets. Moreover, RT-PCR readily detects low-abundance transcripts from this RNA. The Absolutely RNA RT-PCR miniprep kit effectively generates high-purity, intact total RNA from a range of tissues and cultured cells, ready for the most sensitive RT-PCR experiments.2
The ProSTAR first strand RT-PCR kit was used for cDNA synthesis.3 For GAPDH primer detection of DNA in total RNA samples, control cDNA synthesis reactions were performed in the absence of reverse transcriptase. PCR reactions contained 1 l from the cDNA reactions (100 ng of total RNA), 200 M of each dNTP, 25 ng of each GAPDH primer, 1X Taq polymerase buffer, and 1.25 U of Taq2000 DNA polymerase. PCR reactions were performed in a RoboCycler 96 temperature cycler, starting with a 1-minute incubation at 95C and followed by 30 cycles of 95C for 30 seconds, 55C for 1 minute, and 72C for 30 seconds.
For PCR to detect transcripts of varying abundance levels, Taq2000 DNA polymerase was used according to instructions provided with the primers. For each primer set in the PCR reactions, 1 l of cDNA was used.