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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.


Inhibition of RI activity by a monoclonal Ab specific to RI (3F11). (A) 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), 1 ng of RNase A, 40 units of human placental RI (hpRI), or a combination of the above. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis. An asterisk (⁎) indicates that the RNA concentration was too low for calculation of RQI. (B) A similar experiment as shown in (A) was performed using CL Buffer supplemented with 1 mM dithiothreitol (DTT).
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f0010: Inhibition of RI activity by a monoclonal Ab specific to RI (3F11). (A) 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), 1 ng of RNase A, 40 units of human placental RI (hpRI), or a combination of the above. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis. An asterisk (⁎) indicates that the RNA concentration was too low for calculation of RQI. (B) A similar experiment as shown in (A) was performed using CL Buffer supplemented with 1 mM dithiothreitol (DTT).

Mentions: Purified total RNA from Vero cells (5 µg in 200 µL of CL Buffer) was mixed with 1 µg (1 µL) of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9), 1 ng (1 µL) of RNase A (Qiagen; 19101; diluted in CL Buffer), 40 units (1 µL) of human placental RI (hpRI; New England Biolabs; M0307), or a combination of the above; in reactions containing hpRI, its addition preceded the addition of other components. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following the incubation, RNA was purified and subjected to Experion analysis. Data from this experiment are shown in Fig. 2A. The nuclease activity associated with 1 ng of RNase A (lane 4) exceeded the contaminating nuclease activity associated with the monoclonal Ab reagents (lanes 2 and 3). The functionality of hpRI was demonstrated by protection against RNA degradation mediated by RNase A at 22 °C incubation for 1 h (lane 5); further addition of GAPDH Ab did not affect RNA stability (lane 6), whereas addition of RI Ab led to substantial RNA degradation (lane 7). At the more stringent stress condition (37 °C for 1 h), hpRI was not able to protect RNA from degradation (lanes 10 and 11), likely due to the instability of RI in the absence of reducing agent [3].


Data on the inhibition of RNase inhibitor activity by a monoclonal antibody as assessed by microfluidics-based RNA electrophoresis
Inhibition of RI activity by a monoclonal Ab specific to RI (3F11). (A) 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), 1 ng of RNase A, 40 units of human placental RI (hpRI), or a combination of the above. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis. An asterisk (⁎) indicates that the RNA concentration was too low for calculation of RQI. (B) A similar experiment as shown in (A) was performed using CL Buffer supplemented with 1 mM dithiothreitol (DTT).
© Copyright Policy - CC BY
Related In: Results  -  Collection

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f0010: Inhibition of RI activity by a monoclonal Ab specific to RI (3F11). (A) 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), 1 ng of RNase A, 40 units of human placental RI (hpRI), or a combination of the above. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following incubation, RNA was purified and subjected to Experion analysis. An asterisk (⁎) indicates that the RNA concentration was too low for calculation of RQI. (B) A similar experiment as shown in (A) was performed using CL Buffer supplemented with 1 mM dithiothreitol (DTT).
Mentions: Purified total RNA from Vero cells (5 µg in 200 µL of CL Buffer) was mixed with 1 µg (1 µL) of monoclonal Ab specific to either RI (Origene; TA501875; clone 3F11) or GAPDH (Origene; TA802519; clone 2D9), 1 ng (1 µL) of RNase A (Qiagen; 19101; diluted in CL Buffer), 40 units (1 µL) of human placental RI (hpRI; New England Biolabs; M0307), or a combination of the above; in reactions containing hpRI, its addition preceded the addition of other components. The mixtures were incubated for 1 h at 22 °C or at 37 °C. Following the incubation, RNA was purified and subjected to Experion analysis. Data from this experiment are shown in Fig. 2A. The nuclease activity associated with 1 ng of RNase A (lane 4) exceeded the contaminating nuclease activity associated with the monoclonal Ab reagents (lanes 2 and 3). The functionality of hpRI was demonstrated by protection against RNA degradation mediated by RNase A at 22 °C incubation for 1 h (lane 5); further addition of GAPDH Ab did not affect RNA stability (lane 6), whereas addition of RI Ab led to substantial RNA degradation (lane 7). At the more stringent stress condition (37 °C for 1 h), hpRI was not able to protect RNA from degradation (lanes 10 and 11), likely due to the instability of RI in the absence of reducing agent [3].

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.