At Ambion, we have found that oligo d(T) selection can even change the apparent abundance of transcripts from genes that are thought to have invariant expression. For example, when we compare the relative enrichment of cyclophilin and GAPDH transcripts by Northern blot analysis of total versus oligo d(T) selected mouse RNA, we see an obvious change in the apparent abundance of these two transcripts. As shown in Figure 2, oligo d(T) selection enriches GAPDH and cyclophilin 17X and 22X, respectively, from kidney RNA, but 21X and 28X from thymus RNA. The source of this variation is unclear, but the implications for quantitation from oligo d(T) selected RNA are impossible to ignore.
| Figure 2. Differential Enrichment of Specific mRNAs by Oligo dT Chromatography. A Northern blot containing total RNA (1 µg) and twice oligo d(T) selected RNA (50 ng) from mouse thymus and kidney was hybridized simultaneously with GAPDH and cyclophilin RNA probes. Hybridization signals were quantitated with a Bio-Rad Molecular Imager. |
Primer Design
Primers for quantitative experiments are typically designed to amplify a target between 150 and 600 base pairs. Targets smaller than 200 bp are difficult to resolve on agarose gels, and larger targets place a greater burden on the investigator to optimize PCR conditions. The critical aspect for RT-PCR primer choice with respect to minimizing the problems associated with DNA contamination is to design primers that span at least one intron of the genomic sequence. This will result in a PCR product from genomic contamination that will be larger in size than the product generated from the cDNA. In fact, primers can be designed to span a sufficiently large genomic fragment such that amplification from contaminating DNA may be not be possible. For genes in which the genomic sequence is published, the positions of the splice junctions can be found by retrieving the sequence from the Genbank database at http://www.ncbi.nlm.nih.gov/Genbank/index.html. If the intron - exon structure is unknown, primers can be synthesized in different regions of the cDNA sequence and tried in combinations on both cDNA and genomic DNA. It should be possible to choose a primer combination that yields either no product (additional intron sequence produces too long a target for efficient PCR) or an easily distinguishable product when amplifying from genomic DNA. An additional wrinkle is that pseudogenes exist in the mammalian genome for many genes, including the most commonly used internal controls (ß-actin, GAPDH, cyclophilin). These sequences, arising from integration of a reverse transcription product into the genome, do not have introns. Thus, the size of a PCR product amplified from a pseudogene may be identical to that produced from a cDNA copy. The only way to identify these products is to perform a "no-RT" control as shown in Figure 3. The true product from RNA is 361 base pairs. The "no-RT" control yields both a fragment identical in size to the expected RT-PCR product from the RNA transcript (from a pseudogene), and a 1.2 kb fragment from the legitimate genomic locus. If it is absolutely essential to avoid amplification from these sequences, an amplified fragment from a pseudogene may be sequenced, and primers designed to regions where the sequence varies from the legitimate copy of the gene. As little as a one or two nucleotide difference at the 3' end of a primer binding site can completely inhibit amplification from the pseudogene
Figure 3. DNA Contamination in RNA.
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