Identification of HPRT1 PGs for primer design.

<p><b>Top panel</b>: Blat search using HPRT1 mRNA sequence (1405-bp long after deletion of the poly-A tail) as the bait pulls out three putative PGs, besides the authentic HPRT1 genomic sequence that spans 40514 nt on the plus strand of X chromosome. The three putative PGs match the 193–1383rd, the 269–1413th, and the 213–1143rd nt regions of the HPRT1 mRNA as illustrated. There are three additional very short fragments, spanning only 28, 35 and 35 bp, respectively, that also match parts of HPRT1 mRNA but are not considered as PGs. <b>Bottom panel</b>: We pulled out the sequence of each PG (by clicking “details”) and assembled those homologous parts to construct the “cDNA” of the PGs on chromosomes 11 and 4. Alignment of the three sequences with the HPRT1 mRNA reveals that the HPRT1 mRNA has some unique regions. The forward and reverse primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-t002" target="_blank">table 2</a>) we designed are underlined.</p

Alignment of the Actb mRNA with the six PGs that are the most homologous to the Actb mRNA shows that the Actb mRNA has no unique region that is long enough to be a primer.

<p>The 1471–1489th nt and 1852–1870th nt regions of the Actb mRNA (underlined) have the most mismatches compared with other regions and thus are used as the forward and reverse primers, respectively.</p

Alignment of the ACTB mRNA with six PGs that are the most homologous to the ACTB mRNA shows that the ACTB mRNA (after deletion of the poly-A tail) has no unique region that is long enough to be a primer.

<p>The 1452–1473rd nt and the 1678–1697th nt regions of the ACTB mRNA (underlined) encompass the most mismatches compared with other regions, and thus are selected as the forward and reverse primers, respectively.</p

Pseudogenes as Weaknesses of ACTB (Actb) and GAPDH (Gapdh) Used as Reference Genes in Reverse Transcription and Polymerase Chain Reactions

<div><p>The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA.</p> </div

Alignment of the human GAPDH mRNA with seven PGs that are the best homologous to the wild type GAPDH mRNA shows that the first 26 nt of the mRNA is the only unique region while the 685–705th nt region of the GAPDH has the most mismatches (shown as underlined lowercase letters in PGs).

<p>We select the 6–25th and the 685–705th regions as the forward and reverse primers (underlined), respectively.</p

Number of putative pseudogenes.

<p>Note: Only those putative pseudogenes that score over 200 are counted, with details are presented in S-<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g002" target="_blank">Fig. 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g004" target="_blank">4</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-g005" target="_blank">5</a>.</p

Determination of the primer specificity for PCR both vertically and horizontally.

<p><b>A–C</b>: gDNA (G) and cDNA (C) samples from a panel of human cell lines (described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#s2" target="_blank">materials and methods</a>) were amplified by PCR with conditions of initial denature at 95°C for 5 min and 40 cycles of melting at 95°C for 30 sec, primer-annealing at 58°C for 30 sec, and elongating at 72°C for 30 sec. The reaction was determined at 72°C for 10 min. M is molecular weight marker. <b>D</b> and <b>E</b>: gDNA (G) and cDNA (C) samples from M8 and ND5 mouse cell lines were amplified by PCR under the conditions described above. Stars indicate the authentic Actb cDNA band whereas arrows indicate its counterpart from gDNA samples.</p

Primer information.

<p>Note: The “h” or “m” in front of each primer's name indicates the human or mouse origin. “F” or “R” indicates a forward or reverse primer. The number after “F” indicates the position of the first nucleotide (nt) of that primer in the mRNA sequence, whereas the number after “R” indicates the position of the last nt of that primer in the mRNA sequence. Thus, the “R” number minuses the “F” number and then pluses one is the size of the DNA fragment amplified by PCR.</p

Alignment of the mouse Gapdh mRNA with seven PGs that are the best homologous to the Gapdh mRNA shows that these PGs are almost identical to the Gapdh with only several mismatches.

<p>No region of the Gapdh can be used as a primer that can significantly discriminate against any of the PGs.</p

Identification of Hprt PG for primer design.

<p><b>Top panel</b>: Blat search using Hprt mRNA sequence as the bait pulls out only one putative PG, besides the authentic Hprt genomic sequence that spans 33583 bp in the plus strand of the mouse X chromosome. This PG matches the 240–1248th nt of the Hprt mRNA and spans 933 nt on the mouse chromosome 17. <b>Bottom panel</b>: We pulled out the PG sequence and assembled the parts that are homologous to the Hprt mRNA to construct a cDNA. Alignment of the assembled cDNA with the Hprt mRNA reveals that the Hprt mRNA has several unique regions. The forward and reverse primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041659#pone-0041659-t002" target="_blank">table 2</a>) we designed in some unique regions are underlined.</p