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Small RNA existed in commercial reverse transcriptase: primary evidence of functional small RNAs.

Xu J, Chen X, Li D, Chen Q, Zhou Z, Hou D, Wang J, Zhang Q, Zen K, Zhang CY - Protein Cell (2015)

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China.

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MicroRNAs (miRNAs) are small RNA molecules (approximately 22 nucleotides long) that take part in the post-transcriptional regulation of gene expression (Chen and Rajewsky, )... These small molecules have been shown to play an important regulatory role in a wide range of biological and pathological processes (Brennecke et al., ; Cuellar and McManus, ; Lim et al., )... Recently, miRNAs have been found in serum, plasma, urine and other bodily fluids (Chen et al., ; Weber et al., )... Interestingly, the results showed a dose-dependent pattern in the RT-qPCR reaction (Fig.  1B)... The CT values of these miRNAs were found in almost the same Log2 proportions as the volumes of reverse transcriptase... These results implied that the miRNAs found in AMV reverse transcriptase might explain the high background signals... As shown in Fig.  1D, four miRNAs were detected in different commercial AMV reverse transcriptases, and the miRNA expression profiles from different companies were diverse... We also detected other miRNAs and found that different AMV reverse transcriptases have slightly different miRNA contents... However, we wondered whether small RNAs from E. coli would contaminate the M-MLV reverse transcriptase, so we extracted the RNA from commercial M-MLV (Co. 1) and performed Solexa sequencing... We found a large quantity of small RNAs (under 50 nucleotides) in the M-MLV reverse transcriptase... The above results indicated that commercial M-MLV reverse transcriptase also contains small RNAs derived from their artificial E. coli host... According to our results, both AMV and M-MLV reverse transcriptases contain small RNA fragments coming from their host, respectively... Interestingly, Solexa sequencing detected a large number of small RNAs (approximately 20–25 nucleotides) that matched the E. coli genome.

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MiRNAs exists in purified AMV reverse transcriptase. (A) Four miRNAs reverse transcribed with AMV but not M-MLV from different companies exhibited high background in no-template-control. (B) AMV with higher concentration contained more miRNAs. (C) Solexa sequencing revealed large copy numbers of miRNAs in AMV. (D) MiRNAs expression was found variously in AMV from different companies. (E) and (F) The miRNA profile for chick plasma was consistent with that of the reverse transcriptase (R2 = 0.90 and P value = 0.0003)
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Fig1: MiRNAs exists in purified AMV reverse transcriptase. (A) Four miRNAs reverse transcribed with AMV but not M-MLV from different companies exhibited high background in no-template-control. (B) AMV with higher concentration contained more miRNAs. (C) Solexa sequencing revealed large copy numbers of miRNAs in AMV. (D) MiRNAs expression was found variously in AMV from different companies. (E) and (F) The miRNA profile for chick plasma was consistent with that of the reverse transcriptase (R2 = 0.90 and P value = 0.0003)

Mentions: In practice, DEPC-treated water or ultrapure water (Invitrogen) served as no-template-controls for miRNA quantitation. However, certain miRNAs (for example, Let-7a, miR-16, miR-26a, and miR-191 in this study) exhibited high background signals in these no-template-controls when AMV reverse transcriptase was used (Fig. 1A). DNA sequencing showed that the PCR products were correct (data not shown). We tried two other commercial AMV reverse transcriptases (purchased from company 2, Co. 2 and company 3, Co. 3) and M-MLV reverse transcriptase (purchased from Co. 1). These miRNAs could also be detected in all AMV reverse transcriptase, but not in M-MLV reverse transcriptase (Fig. 1A). The above results indicated that the miRNA templates were inserted during reverse transcription when using the AMV reverse transcriptase system. Therefore, we tested all the reagents used during RT-qPCR. Our experiments excluded the possibility of miRNA contamination in all the reagents except the AMV reverse transcriptase (data not shown). In fact, only the reverse transcriptase is derived from biologically active substances, which is likely being contaminated during production. We extracted the total RNA from 10 μL or 160 μL of AMV reverse transcriptase (Co. 1), and the total RNA samples served as templates for the reverse transcription reaction; M-MLV reverse transcriptase was used because of its low background. Interestingly, the results showed a dose-dependent pattern in the RT-qPCR reaction (Fig. 1B). The CT values of these miRNAs were found in almost the same Log2 proportions as the volumes of reverse transcriptase. These results implied that the miRNAs found in AMV reverse transcriptase might explain the high background signals.Figure 1


Small RNA existed in commercial reverse transcriptase: primary evidence of functional small RNAs.

Xu J, Chen X, Li D, Chen Q, Zhou Z, Hou D, Wang J, Zhang Q, Zen K, Zhang CY - Protein Cell (2015)

MiRNAs exists in purified AMV reverse transcriptase. (A) Four miRNAs reverse transcribed with AMV but not M-MLV from different companies exhibited high background in no-template-control. (B) AMV with higher concentration contained more miRNAs. (C) Solexa sequencing revealed large copy numbers of miRNAs in AMV. (D) MiRNAs expression was found variously in AMV from different companies. (E) and (F) The miRNA profile for chick plasma was consistent with that of the reverse transcriptase (R2 = 0.90 and P value = 0.0003)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4286138&req=5

Fig1: MiRNAs exists in purified AMV reverse transcriptase. (A) Four miRNAs reverse transcribed with AMV but not M-MLV from different companies exhibited high background in no-template-control. (B) AMV with higher concentration contained more miRNAs. (C) Solexa sequencing revealed large copy numbers of miRNAs in AMV. (D) MiRNAs expression was found variously in AMV from different companies. (E) and (F) The miRNA profile for chick plasma was consistent with that of the reverse transcriptase (R2 = 0.90 and P value = 0.0003)
Mentions: In practice, DEPC-treated water or ultrapure water (Invitrogen) served as no-template-controls for miRNA quantitation. However, certain miRNAs (for example, Let-7a, miR-16, miR-26a, and miR-191 in this study) exhibited high background signals in these no-template-controls when AMV reverse transcriptase was used (Fig. 1A). DNA sequencing showed that the PCR products were correct (data not shown). We tried two other commercial AMV reverse transcriptases (purchased from company 2, Co. 2 and company 3, Co. 3) and M-MLV reverse transcriptase (purchased from Co. 1). These miRNAs could also be detected in all AMV reverse transcriptase, but not in M-MLV reverse transcriptase (Fig. 1A). The above results indicated that the miRNA templates were inserted during reverse transcription when using the AMV reverse transcriptase system. Therefore, we tested all the reagents used during RT-qPCR. Our experiments excluded the possibility of miRNA contamination in all the reagents except the AMV reverse transcriptase (data not shown). In fact, only the reverse transcriptase is derived from biologically active substances, which is likely being contaminated during production. We extracted the total RNA from 10 μL or 160 μL of AMV reverse transcriptase (Co. 1), and the total RNA samples served as templates for the reverse transcription reaction; M-MLV reverse transcriptase was used because of its low background. Interestingly, the results showed a dose-dependent pattern in the RT-qPCR reaction (Fig. 1B). The CT values of these miRNAs were found in almost the same Log2 proportions as the volumes of reverse transcriptase. These results implied that the miRNAs found in AMV reverse transcriptase might explain the high background signals.Figure 1

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, 210093, China.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

MicroRNAs (miRNAs) are small RNA molecules (approximately 22 nucleotides long) that take part in the post-transcriptional regulation of gene expression (Chen and Rajewsky, )... These small molecules have been shown to play an important regulatory role in a wide range of biological and pathological processes (Brennecke et al., ; Cuellar and McManus, ; Lim et al., )... Recently, miRNAs have been found in serum, plasma, urine and other bodily fluids (Chen et al., ; Weber et al., )... Interestingly, the results showed a dose-dependent pattern in the RT-qPCR reaction (Fig.  1B)... The CT values of these miRNAs were found in almost the same Log2 proportions as the volumes of reverse transcriptase... These results implied that the miRNAs found in AMV reverse transcriptase might explain the high background signals... As shown in Fig.  1D, four miRNAs were detected in different commercial AMV reverse transcriptases, and the miRNA expression profiles from different companies were diverse... We also detected other miRNAs and found that different AMV reverse transcriptases have slightly different miRNA contents... However, we wondered whether small RNAs from E. coli would contaminate the M-MLV reverse transcriptase, so we extracted the RNA from commercial M-MLV (Co. 1) and performed Solexa sequencing... We found a large quantity of small RNAs (under 50 nucleotides) in the M-MLV reverse transcriptase... The above results indicated that commercial M-MLV reverse transcriptase also contains small RNAs derived from their artificial E. coli host... According to our results, both AMV and M-MLV reverse transcriptases contain small RNA fragments coming from their host, respectively... Interestingly, Solexa sequencing detected a large number of small RNAs (approximately 20–25 nucleotides) that matched the E. coli genome.

Show MeSH