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Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis

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ABSTRACT

Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled “Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)” (Wang et al., 2016) [1].

No MeSH data available.


Contaminating RNase activity in Ab reagents. Purified total RNA (5 µg in 200 µL of CL Buffer) was mixed with 1 µg of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9). The mixtures were incubated for 1 h on ice, at 22 °C, or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis.
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f0005: Contaminating RNase activity in Ab reagents. Purified total RNA (5 µg in 200 µL of CL Buffer) was mixed with 1 µg of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9). The mixtures were incubated for 1 h on ice, at 22 °C, or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis.

Mentions: Data from this experiment are shown in Fig. 1. The extent of RNase contamination was comparable between the two Ab reagents; according to information from the supplier, both were purified from mouse ascites fluid by affinity chromatography. RNA in control reactions in the absence of Ab (CL Buffer alone) was intact even after incubation for 1 h at 37 °C (RQI of 9.7), thereby demonstrating that our CL Buffer components are free of RNase activity.


Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis
Contaminating RNase activity in Ab reagents. Purified total RNA (5 µg in 200 µL of CL Buffer) was mixed with 1 µg of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9). The mixtures were incubated for 1 h on ice, at 22 °C, or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037242&req=5

f0005: Contaminating RNase activity in Ab reagents. Purified total RNA (5 µg in 200 µL of CL Buffer) was mixed with 1 µg of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9). The mixtures were incubated for 1 h on ice, at 22 °C, or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis.
Mentions: Data from this experiment are shown in Fig. 1. The extent of RNase contamination was comparable between the two Ab reagents; according to information from the supplier, both were purified from mouse ascites fluid by affinity chromatography. RNA in control reactions in the absence of Ab (CL Buffer alone) was intact even after incubation for 1 h at 37 °C (RQI of 9.7), thereby demonstrating that our CL Buffer components are free of RNase activity.

View Article: PubMed Central - PubMed

ABSTRACT

Using purified reaction components, a commercial monoclonal antibody (Ab) specific to RNase inhibitor (RI) was found to interfere with the activity of RI. Total RNA was mixed with a monoclonal Ab specific to either RI (clone 3F11) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNase A, RI, or a combination of the above. Following incubation for 1 h at 22 °C or 37 °C, RNA integrity of the mixtures was assessed using microfluidics-based Bio-Rad Experion RNA electrophoresis. The addition of Ab 3F11 prevented RI from effectively inhibiting RNase A and therefore resulted in extensive RNA degradation. The data presented are associated with the research article entitled “Endogenous RNase Inhibitor Contributes to Stability of RNA in Crude Cell Lysates: Applicability to Reverse Transcription Quantitative PCR (RT-qPCR)” (Wang et al., 2016) [1].

No MeSH data available.