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Short RNA indicator sequences are not completely degraded by autoclaving.

Unnithan VV, Unc A, Joe V, Smith GB - Sci Rep (2014)

Bottom Line: Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification.Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses.If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, New Mexico State University, Las Cruces, NM, USA, 88003.

ABSTRACT
Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

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Quantification and quality control steps for three amplicons that were consistently recovered after repeated autoclaving.(A), (B) and (C) indicate amplicons commonly employed as indicator sequences for the presence and quantification of MS2 coliphage7. Numeric subscripts indicate the number of autoclave cycles (e.g. A1, B1, C1). If no numeric subscript is present then the capital letter indicates results obtained from non-autoclaved positive controls. 1.i. The horizontal line represents the threshold Cq 40. The quantification cycles of A2 & A3, B2 & B3 and C2 & C3 are overlapping. 1.iv. Plasmid extracts are indicated by the subscript “p” (e.g. pA1, pB1, pC1). 1.v. is a paired comparison of A1, B1, C1 (bottom sequence in each pair) to A, B, C (top sequence in each pair). The first two pairs are sequenced in the 3′-5′ direction and the third pair in the 5′-3′ direction. The gels shown in this image have been cropped for convenience. The borders between separate gels have been marked with a black line.
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f1: Quantification and quality control steps for three amplicons that were consistently recovered after repeated autoclaving.(A), (B) and (C) indicate amplicons commonly employed as indicator sequences for the presence and quantification of MS2 coliphage7. Numeric subscripts indicate the number of autoclave cycles (e.g. A1, B1, C1). If no numeric subscript is present then the capital letter indicates results obtained from non-autoclaved positive controls. 1.i. The horizontal line represents the threshold Cq 40. The quantification cycles of A2 & A3, B2 & B3 and C2 & C3 are overlapping. 1.iv. Plasmid extracts are indicated by the subscript “p” (e.g. pA1, pB1, pC1). 1.v. is a paired comparison of A1, B1, C1 (bottom sequence in each pair) to A, B, C (top sequence in each pair). The first two pairs are sequenced in the 3′-5′ direction and the third pair in the 5′-3′ direction. The gels shown in this image have been cropped for convenience. The borders between separate gels have been marked with a black line.

Mentions: After the 1st autoclave cycle plaque assays were negative, confirming inactivation of MS2. However three commonly used indicator primer sets7 (listed here as A, B and C, Figure 1 and Supplementary materials), producing amplicons in the 70 to 77 bp range, permitted qRT PCR quantification at 0.18%, 0.015% and 0.009% respectively, of the original concentration. After the 2nd and 3rd autoclave cycles the genetic material degraded such that qRT PCR quantification could only be obtained after more than 35 quantification cycles (Cq) (Figure 1.i.). Primer set A targeted the RNA replicase β chain while primer sets B and C targeted assembly proteins on the RNA genome of MS2 bacteriophage7.


Short RNA indicator sequences are not completely degraded by autoclaving.

Unnithan VV, Unc A, Joe V, Smith GB - Sci Rep (2014)

Quantification and quality control steps for three amplicons that were consistently recovered after repeated autoclaving.(A), (B) and (C) indicate amplicons commonly employed as indicator sequences for the presence and quantification of MS2 coliphage7. Numeric subscripts indicate the number of autoclave cycles (e.g. A1, B1, C1). If no numeric subscript is present then the capital letter indicates results obtained from non-autoclaved positive controls. 1.i. The horizontal line represents the threshold Cq 40. The quantification cycles of A2 & A3, B2 & B3 and C2 & C3 are overlapping. 1.iv. Plasmid extracts are indicated by the subscript “p” (e.g. pA1, pB1, pC1). 1.v. is a paired comparison of A1, B1, C1 (bottom sequence in each pair) to A, B, C (top sequence in each pair). The first two pairs are sequenced in the 3′-5′ direction and the third pair in the 5′-3′ direction. The gels shown in this image have been cropped for convenience. The borders between separate gels have been marked with a black line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921633&req=5

f1: Quantification and quality control steps for three amplicons that were consistently recovered after repeated autoclaving.(A), (B) and (C) indicate amplicons commonly employed as indicator sequences for the presence and quantification of MS2 coliphage7. Numeric subscripts indicate the number of autoclave cycles (e.g. A1, B1, C1). If no numeric subscript is present then the capital letter indicates results obtained from non-autoclaved positive controls. 1.i. The horizontal line represents the threshold Cq 40. The quantification cycles of A2 & A3, B2 & B3 and C2 & C3 are overlapping. 1.iv. Plasmid extracts are indicated by the subscript “p” (e.g. pA1, pB1, pC1). 1.v. is a paired comparison of A1, B1, C1 (bottom sequence in each pair) to A, B, C (top sequence in each pair). The first two pairs are sequenced in the 3′-5′ direction and the third pair in the 5′-3′ direction. The gels shown in this image have been cropped for convenience. The borders between separate gels have been marked with a black line.
Mentions: After the 1st autoclave cycle plaque assays were negative, confirming inactivation of MS2. However three commonly used indicator primer sets7 (listed here as A, B and C, Figure 1 and Supplementary materials), producing amplicons in the 70 to 77 bp range, permitted qRT PCR quantification at 0.18%, 0.015% and 0.009% respectively, of the original concentration. After the 2nd and 3rd autoclave cycles the genetic material degraded such that qRT PCR quantification could only be obtained after more than 35 quantification cycles (Cq) (Figure 1.i.). Primer set A targeted the RNA replicase β chain while primer sets B and C targeted assembly proteins on the RNA genome of MS2 bacteriophage7.

Bottom Line: Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification.Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses.If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, New Mexico State University, Las Cruces, NM, USA, 88003.

ABSTRACT
Short indicator RNA sequences (<100 bp) persist after autoclaving and are recovered intact by molecular amplification. Primers targeting longer sequences are most likely to produce false positives due to amplification errors easily verified by melting curves analyses. If short indicator RNA sequences are used for virus identification and quantification then post autoclave RNA degradation methodology should be employed, which may include further autoclaving.

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