New Study Discovers Significant RT-qPCR Artifacts in CRISPR Research and Introduces UltraMarathonRT® as a Solution
A recent study conducted by researchers at Johns Hopkins University has uncovered a significant issue regarding reverse transcription quantitative polymerase chain reaction (RT-qPCR) artifacts prevalent in CRISPR knockdown studies. Published in Nature Biotechnology, the research indicates a critical flaw in conventional RT-qPCR kits that may inflate the perceived knockdown (KD) efficiency.
The study focused on RNA-targeting CRISPR systems, particularly the Csm system, examining how a catalytically inactive version of the enzyme still yielded a strong apparent knockdown signal. This occurred despite confirming that neither RNA cleavage nor protein-level knockdown was taking place. The researchers traced this unexpected signal back to the RT-qPCR kits employed in their assays. Traditional retroviral reverse transcriptases, such as MMLV RT, are commonly used to convert RNA into complementary DNA (cDNA) during this process. However, these enzymes are characterized by a lack of strand displacement capability, which results in issues when they encounter tightly bound guide RNA (gRNA).
The study revealed that the continually bound gRNA effectively obstructs the reverse transcriptase from accurately synthesizing cDNA, thereby suppressing any RT-qPCR amplicons situated upstream of the gRNA binding site. Consequently, this led to falsely inflated knockdown efficiency conclusions. This issue was consistently observed across various RNA-targeting CRISPR systems, including PspCas13b and CasRx. The researchers estimated that this artifact is likely widespread in the literature, given that cut-spanning RT-qPCR is a favored method for quantifying RNA in these experiments.
However, the researchers were able to identify a straightforward approach to rectify this quantification issue. As the problem originates during reverse transcription rather than being a biological interaction, the use of a highly efficient, strand-displacing reverse transcriptase led to the resolution of the artifact. By utilizing UltraMarathonRT (uMRT), which is an enzyme derived from group II introns, the team observed no apparent knockdown signals in any of the RNase-dead controls, irrespective of the position of the amplicon tested. This confirmed uMRT's capability to bypass the impediment posed by the bound gRNA.
Leslie Watkins, a member of the Johns Hopkins research team, remarked, "We did not initially aim to discover this quantification artifact, but our findings now illustrate that misleading measurements have likely skewed reported knockdown efficiencies across the field. Conventional RT kits aren't as reliable as previously believed, even by myself. UltraMarathonRT provided just the solution we required to obtain accurate measurements and advance our technologies towards practical research and therapeutic applications."
Furthermore, uMRT demonstrated consistent, orthogonally-validated knockdown values that remained consistent regardless of where the amplicon was placed in the process. This has helped restore researchers' confidence in RT-qPCR assays that are extensively used for RNA-targeting CRISPR knockouts and knockdowns.
The discovery was welcomed by many in the scientific community, with Jason Underwood, the Vice President of Technology Development at RNAConnect, noting, "It is remarkable to see how a standard control experiment could evolve into such a significant discovery for the entire RNA-targeting CRISPR domain. Our study definitively establishes that gRNA remains attached to its target mRNA following RNA preparation, which halts retroviral reverse transcriptases. We are delighted to find that UltraMarathonRT's impressive strand-displacement function effectively addressed this challenge and provided precise quantification."
RNAConnect, the company behind UltraMarathonRT, is a life science firm located in Branford, Connecticut, dedicated to offering advanced tools for the RNA research sector. Their UltraMarathonRT products enhance cDNA synthesis, RT-PCR, RT-qPCR, and RNA sequencing workflows, significantly raising detection and quantification standards for complex RNA transcripts.
For additional information on RNAConnect and its cutting-edge products, visit their website at www.rnaconnect.com. This research not only sheds light on existing flaws within the CRISPR research framework but also paves the way for enhanced accuracy and reliability in future studies.
The study focused on RNA-targeting CRISPR systems, particularly the Csm system, examining how a catalytically inactive version of the enzyme still yielded a strong apparent knockdown signal. This occurred despite confirming that neither RNA cleavage nor protein-level knockdown was taking place. The researchers traced this unexpected signal back to the RT-qPCR kits employed in their assays. Traditional retroviral reverse transcriptases, such as MMLV RT, are commonly used to convert RNA into complementary DNA (cDNA) during this process. However, these enzymes are characterized by a lack of strand displacement capability, which results in issues when they encounter tightly bound guide RNA (gRNA).
The study revealed that the continually bound gRNA effectively obstructs the reverse transcriptase from accurately synthesizing cDNA, thereby suppressing any RT-qPCR amplicons situated upstream of the gRNA binding site. Consequently, this led to falsely inflated knockdown efficiency conclusions. This issue was consistently observed across various RNA-targeting CRISPR systems, including PspCas13b and CasRx. The researchers estimated that this artifact is likely widespread in the literature, given that cut-spanning RT-qPCR is a favored method for quantifying RNA in these experiments.
However, the researchers were able to identify a straightforward approach to rectify this quantification issue. As the problem originates during reverse transcription rather than being a biological interaction, the use of a highly efficient, strand-displacing reverse transcriptase led to the resolution of the artifact. By utilizing UltraMarathonRT (uMRT), which is an enzyme derived from group II introns, the team observed no apparent knockdown signals in any of the RNase-dead controls, irrespective of the position of the amplicon tested. This confirmed uMRT's capability to bypass the impediment posed by the bound gRNA.
Leslie Watkins, a member of the Johns Hopkins research team, remarked, "We did not initially aim to discover this quantification artifact, but our findings now illustrate that misleading measurements have likely skewed reported knockdown efficiencies across the field. Conventional RT kits aren't as reliable as previously believed, even by myself. UltraMarathonRT provided just the solution we required to obtain accurate measurements and advance our technologies towards practical research and therapeutic applications."
Furthermore, uMRT demonstrated consistent, orthogonally-validated knockdown values that remained consistent regardless of where the amplicon was placed in the process. This has helped restore researchers' confidence in RT-qPCR assays that are extensively used for RNA-targeting CRISPR knockouts and knockdowns.
The discovery was welcomed by many in the scientific community, with Jason Underwood, the Vice President of Technology Development at RNAConnect, noting, "It is remarkable to see how a standard control experiment could evolve into such a significant discovery for the entire RNA-targeting CRISPR domain. Our study definitively establishes that gRNA remains attached to its target mRNA following RNA preparation, which halts retroviral reverse transcriptases. We are delighted to find that UltraMarathonRT's impressive strand-displacement function effectively addressed this challenge and provided precise quantification."
RNAConnect, the company behind UltraMarathonRT, is a life science firm located in Branford, Connecticut, dedicated to offering advanced tools for the RNA research sector. Their UltraMarathonRT products enhance cDNA synthesis, RT-PCR, RT-qPCR, and RNA sequencing workflows, significantly raising detection and quantification standards for complex RNA transcripts.
For additional information on RNAConnect and its cutting-edge products, visit their website at www.rnaconnect.com. This research not only sheds light on existing flaws within the CRISPR research framework but also paves the way for enhanced accuracy and reliability in future studies.
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