Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR

<p>Abstract</p> <p>Background</p> <p>The ability to accurately measure patterns of gene expression is essential in studying gene function. The reverse transcription polymerase chain reaction (RT-PCR) has become the method of choice for the detection and measurement of RNA expression patterns in both cells and small quantities of tissue. Our previous results show that there is a significant production of primer-independent cDNA synthesis using a popular RNase H^-RT enzyme. A PCR product was amplified from RT reactions that were carried out without addition of RT-primer. This finding jeopardizes the accuracy of RT-PCR when analyzing RNA that is expressed in both orientations. Current literature findings suggest that naturally occurring antisense expression is widespread in the mammalian transcriptome and consists of both coding and non-coding regulatory RNA. The primary purpose of this present study was to investigate the occurrence of primer-independent cDNA synthesis and how it may influence the accuracy of detection of sense-antisense RNA pairs.</p> <p>Results</p> <p>Our findings on cellular RNA and <it>in vitro </it>synthesized RNA suggest that these products are likely the results of RNA self-priming to generate random cDNA products, which contributes to the loss of strand specificity. The use of RNase H⁺RT enzyme and carrying the RT reaction at high temperature (50°C) greatly improved the strand specificity of the RT-PCR detection.</p> <p>Conclusion</p> <p>While RT PCR is a basic method used for the detection and quantification of RNA expression in cells, primer-independent cDNA synthesis can interfere with RT specificity, and may lead to misinterpretation of the results, especially when both sense and antisense RNA are expressed. For accurate interpretation of the results, it is essential to carry out the appropriate negative controls.</p

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development

Intergenic bidirectional promoter and cooperative regulation of the IIx and IIb MHC genes in fast skeletal muscle

This study investigated the dynamic regulation of IIx-IIb MHC genes in the fast white medial gastrocnemius (WMG) muscle in response to intermittent resistance exercise training (RE), a model associated with a rapid shift from IIb to IIx expression (11). We investigated the effect of 4 days of RE on the transcriptional activity across the skeletal MHC gene locus in the WMG in female Sprague-Dawley rats. Our results show that RE resulted in significant shifts from IIb to IIx observed at both the pre-mRNA and mRNA levels. An antisense RNA (xII NAT) was detected in the intergenic (IG) region between IIx and IIb, extending across the entire IIx gene and into its promoter. The expression of the xII NAT was positively correlated with IIb pre-mRNA (R = +0.8), and negatively correlated with IIx pre-mRNA (R = −0.8). Transcription mapping of the IIx–IIb IG region revealed the generation of sense IIb and xII NATs from a single promoter region. This bidirectional promoter is highly conserved among species and contains several regulatory elements that may be implicated in its regulation. These results suggest that the IIx and the IIb genes are physically and functionally linked via the bidirectional promoter. In order for the IIx MHC gene to be regulated, a feedback mechanism from the IG xII NAT is needed. In conclusion, the IG bidirectional promoter generating antisense RNA appears to be essential for the coordinated regulation of the skeletal muscle MHC genes during dynamic phenotype shifts

Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR-3

<p><b>Copyright information:</b></p><p>Taken from "Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR"</p><p>http://www.biomedcentral.com/1472-6750/7/21</p><p>BMC Biotechnology 2007;7():21-21.</p><p>Published online 4 May 2007</p><p>PMCID:PMC1876213.</p><p></p>and antisense MYH7 RNA expression in NC and PTU heart total RNA. -P is when the RT step was carried out in absence of primers. +R is when the RT was carried out in presence of reverse primer that is targeting the sense RNA. +F is when the RT was carried out in presence of forward primer that is targeting the antisense RNA. . One Step RT-PCR using the Qiagen kit. Reactions used human brain RNA and PCR targeting either sense or antisense RNA of specific genes shown to be expressed in sense/antisense pairs (SA22, SA24) or in a single form (NC3, NC10). These primers were used previously by Chen et al., [9]. As a further test of specificity, the RT reactions were carried out in absence of primers (-P) or in presence of a non specific primer (+N), complementary to human MYH4 mRNA, MYH 4 is not expressed in the brain and is not related to any of the studied genes. One step RT-PCR (Qiagen kit) used to detect small amount of antisense (AS) MYH 7 RNA in mixed total RNA preparations containing either 100% sense (S) MYH7 RNA, (100:0), or 99:1 and 90:10 of S:AS ratios. Sense MYH7 is derived from soleus muscle total RNA, whereas antisense MYH7 is derived from total RNA in T3-treated rat hearts. Reactions contained 100 ng of total RNA of the specified S:AS composition. RT reaction included either no primers (-P), a forward primer (+F) to target the antisense, and a non-specific primer (+N). For all these reactions in a, b, and c, 100 ng total RNA was used per reaction, and the RT was carried out at 50°C for 30 minutes followed by 10 minutes at 95°C. The PCR was carried out for 30 cycles (a, b, and c) or 35 cycles (c). See Additional file 4 for primer information

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development

Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR-0

<p><b>Copyright information:</b></p><p>Taken from "Potential pitfalls in the accuracy of analysis of natural sense-antisense RNA pairs by reverse transcription-PCR"</p><p>http://www.biomedcentral.com/1472-6750/7/21</p><p>BMC Biotechnology 2007;7():21-21.</p><p>Published online 4 May 2007</p><p>PMCID:PMC1876213.</p><p></p>rdiac MYH7 and MYH6 sense RNA, the lower strand transcribes the antisense MYH7 RNA, which is abundant in normal control hearts [1]. : representative gels obtained from RT-PCR targeting sense and antisense RNA corresponding to the MYH7 gene. RT used RNase Henzyme under manufacturer standard conditions (see methods) in presense of specific primers (+p) or in absence of primers (-p). Bar graph depicting the net signal of MYH7 sense and antisense in each group, net consisting of the difference between +p and -p RT-PCR band intensity. Note that a normal control heart in the rat is associated with abundant relatively MYH7 gene expression (MYH6 gene expression is dominant). Under the PTU condition, MYH7sense RNA expression is increased. Antisense MYH7 RNA is strongly expressed in the normal control heart, based on strong net signal. In PTU heart, the antisense MYH7 RNA is decreased to a very low level. Note the +p product is similar to -p when targeting antisense MYH7 RNA in PTU hearts. Bar graph depicting relative no-primer signal (NP) to the total signal in each group as determined by real time PCR methods. Net MYH7 sense and antisense RNA copy numbers in NC and PTU hearts using real time PCR. Data are means ± SE. N = 6/group. See Additional file 4 for primer information. For both sense and antisense MYH7 targets, end-point PCR (b and c) used 0.2 μl of the cDNA and was performed for 28 cycles. For real time PCR, we used 320 nl cDNA for each sample, and the signal was compared to a standard curve established with a serial dilution of a standard consisting of purified PCR product as explained in the methods. See Additional file 4 for primer information. Based on standard curve linear regression analyses, copies for each target RNA were calculated.+p: a strand specific RT primer was included; -p: RT without primer. Sense is the amplification product of the sense target obtained when the reverse primer was added to the RT reaction. Antisense is the amplification product of the antisense target obtained when the forward primer was included in the RT reaction. In all these reactions, the presence of the no primer product depended on the presence of RNA and the RT enzyme, and was not formed in RT reactions that were carried out in the absence of the reverse transcriptase enzyme