Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA

Lemire, Karissa A.; Rodriguez, Yelitza Y.; McIntosh, Michael T.

doi:10.1186/s12985-016-0552-0

Virology Journal

Table 1 Verification of RNA degradation while maintaining plasmid DNA integrity

From: Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA

Sample ID	Heat	Plasmid	CSFV RNA	SCR	NaOH	HCl	TE	PCR	DNA
1- SCR RNA	x	0 μl	0 μl	26 μl	0 μl	0 μl	260 μl	SCR+, CSF-	NA
2- Plasmid DNA	x	130 μl	0 μl	0 μl	0 μl	0 μl	156 μl	SCR-, CSF-	+
3- SCR RNA/Plasmid DNA	x	130 μl	0 μl	26 μl	0 μl	0 μl	130 μl	SCR+, CSF-	+
4- SCR RNA treated	x	0 μl	0 μl	26 μl	60 μl	60 μl	140 μl	SCR+, CSF-	NA
5- Plasmid DNA treated	x	130 μl	0 μl	0 μl	60 μl	60 μl	36 μl	SCR-, CSF-	+^a
6- SCR RNA/Plasmid DNA treated	x	130 μl	0 μl	26 μl	60 μl	60 μl	10 μl	SCR-, CSF-	+^a
7- CSF RNA	x	0 μl	10 μl	0 μl	0 μl	0 μl	276 μl	SCR-, CSF+	NA
8- CSF RNA/SCR RNA	x	0 μl	10 μl	26 μl	0 μl	0 μl	250 μl	SCR+, CSF+	NA
9- CSF RNA/SCR RNA/ Plasmid DNA	x	130 μl	10 μl	26 μl	0 μl	0 μl	166 μl	SCR+, CSF+	+
10- CSF RNA/Plasmid DNA	x	130 μl	10 μl	0 μl	0 μl	0 μl	140 μl	SCR-, CSF+	+
11- CSF RNA treated	x	0 μl	10 μl	0 μl	60 μl	60 μl	130 μl	SCR-, CSF-	NA
12- CSF RNA/SCR RNA treated	x	0 μl	10 μl	26 μl	60 μl	60 μl	156 μl	SCR-, CSF-	NA
13- CSF RNA/SCR RNA/ Plasmid DNA treated	x	130 μl	10 μl	26 μl	60 μl	60 μl	0 μl	SCR-, CSF-	+^a
14- CSF RNA/Plasmid DNA treated	x	130 μl	10 μl	0 μl	60 μl	60 μl	260 μl	SCR-, CSF-	+^a
15- SCR RNA post pH normalization	x	0 μl	0 μl	26μl^b	60 μl	60 μl	146 μl	SCR+, CSF-	NA
16- Plasmid DNA post pH normalization	x	130 μl^b	0 μl	0 μl	60 μl	60 μl	36 μl	SCR-, CSF-	+
17- CSF RNA post pH normalization	x	0 μl	10 μl^b	0 μl	60 μl	60 μl	130 μl	SCR-, CSF+	NA
18- CSF RNA/SCR RNA post pH normalization	x	0 μl	10μl^b	26μl^b	60 μl	60 μl	156 μl	SCR+, CSF+	NA
19- CSF RNA/SCR RNA/Plasmid DNA post pH normalization	x	130μl^b	10μl^b	26μl^b	60 μl	60 μl	0 μl	SCR+, CSF+	+
20- CSF RNA undilute control		0 μl	2.5 μl	0 μl	0 μl	0 μl	0 μl	SCR-, CSF+	NA
21- CSF RNA diluted control		0 μl	10 μl	0 μl	0 μl	0 μl	276 μl	SCR-, CSF+	NA
22- SCR RNA diluted control		0 μl	0 μl	26 μl	0 μl	0 μl	260 μl	SCR+, CSF-	NA
23- SCR RNA undilute control		0 μl	0 μl	2.5 μl	0 μl	0 μl	0 μl	SCR+, CSF-	NA
24- CSF positive amplification control		0 μl	2.5 μl	0 μl	0 μl	0 μl	0 μl	SCR-, CSF+	NA
25- No template control for PCR		0 μl	0 μl	0 μl	0 μl	0 μl	H₂O	SCR-, CSF-	NA

Samples, containing various combinations of SCR RNA, CSF RNA, and pGEM Teasy Vector plasmid, treatment conditions, and results for verification of RNA degradation and maintenance of plasmid DNA integrity are listed. NaOH and heat treatment incubation time used in this initial study was 30 min. ^aSlight nicking or denatured to ssDNA was observed as compared to untreated control plasmid. ^bReagent added post pH neutralization as a positive control sample. NA indicates not analyzed; x indicates 60 °C incubation and 10’ boiling steps were included

Back to article page

ISSN: 1743-422X

Contact us

Submission enquiries: journalsubmissions@springernature.com