Fast and simple fluorescent RNA quality assessment

Transcription

1 Fast and simple fluorescent RNA quality assessment Chris Vonnegut Presented at ASHG Laboratory CoLab Session October, The world leader in serving science

2 RNA Quality and Quantity RNA Sample Isolation Cells Tissue Blood / biological samples Bacteria Plants RNA Quantity & Quality UV-Vis spectroscopy Electrophoresis RNA Sample Analysis qpcr/qrt-pcr RT-PCR RNA-Seq Microarrays Sequencing NGS Cloning RNA quantity & quality measurements Essential steps post-purification

3 Current Techniques for Measuring RNA integrity and quality UV-Absorbance Capillary electrophoresis Gel electrophoresis Pros: Time to result / (protein contamination) / (chemical contamination) No reagents required Cons: Cannot differentiate between intact or degraded RNA No size or distribution info. Pros: Industry standard Size & distribution info. Cons: Time-consuming/tedious workflow Time to result Price per sample Reagent shelf-life Sample # flexibility Pros: Simple Size & distribution info. Cons: Time-consuming / tedious workflow Time to result Sample # flexibility ~ g sample required

4 NEW: Qubit RNA Integrity & Quality (IQ) Assay and Qubit Fluorometer Detect viable from degraded RNA with -dye assay: Unique dye for large and/or structured RNA Unique dye for small, degraded RNA Simple Add RNA sample to Qubit RNA IQ buffer Measure on Qubit Fluorometer Rapid time to result ~ minutes sample preparation ~ seconds per sample

5 Qubit RNA Integrity & Quality (IQ) Assay: RNA Sample Preparation USE: - L of.-. µg RNA Sample NEW: Qubit Reagent Calculator Q: Assays Q: Assays

6 Qubit RNA Integrity & Quality (IQ) Assay: Qubit Fluorometer Workflow Qubit Fluorometer Intuitive Workflow

7 Qubit RNA IQ results RNA IQ#: % small RNA % large and/or structured (mrna, trna, rrna)

8 Selectivity of the RNA IQ reagents for large and/or structured & small RNA. 9. Fluorescence Large RNA Channel Small RNA Channel RNA IQ# ng/ul sirna (Large RNA held constant at ng/ul in sample). % % % 9% % % Large and/or structured RNA Triplicate samples containing ng/ L rrna (E. coli) and varying amounts of sirna ( to ng/ L) were assayed with the Qubit RNA IQ assay on the Qubit Fluorometer.

9 Qubit RNA IQ with solutions containing various amounts of large and small RNA Fluorescence, small RNA channel % % % % % % % % % Large RNA in sample Fluorescence, large RNA Channel RNA IQ 9 % % % % % % % Large RNA Triplicate samples containing a total of ng/ L RNA comprised of small RNA (Silencer Select GAPDH sirna) and large RNA (rrna, E.coli) were assayed with the Qubit RNA IQ assay on the Qubit Fluorometer. 9

10 Real-time RNA degradation monitored with Qubit RNA IQ Assay fm RNase A 9 RNA IQ fm RNase A fm RNase A fm RNase A fm RNase A RFU Time (min) fm RNase A Large and/or structured RNA Small RNA Time (minutes after RNase addition) Time (min) Triplicate samples of ng/ L rrna solutions were incubated with RNase A in the final assay solution. rrna degradation by RNase A was measured in real-time using the assay. RFU Large and/or structured RNA Small RNA

11 RNA assessment by Qubit RNA IQ or Agilent BioAnalyzer following RNase treatment RNA IQ 9 RNA IQ# decreases over time Time (minutes after RNase addition) RIN RIN# decreases over time Time (minutes after RNase addition) Electrophoresis time course Ladder; T=, 9 min fm RNase A was added to aliquots of a ng/ L solution of rrna (E.coli), and at various time points treated with RNaseOUT then measured with either the Qubit RNA IQ Assay with a Qubit Fluorometer or Agilent BioAnalyzer Prokaryote Total RNA Nano chip.

12 RNA measured by BioAnalyzer & Qubit RNA IQ Assay after min. RNase Agilent BioAnalyzer RIN#.9 RNA IQ#: 9. Small RNA: % Large and/or Structured RNA: 9% s rrna & trna rrna was treated with.pg/µl RNase A, followed by the addition of RNaseOUT after minute. Samples were measured either on a Prokaryote Total RNA Nano chip or with the Qubit RNA IQ Assay. Quality scores differences, IQ#: 9. and RIN#:.9 is the result of rrna, mrna & structured s rrna and trna which bind the large and/or structured RNA dye. Peaks in this area can be misinterpreted as degradation in some electrophoretic analyses.

13 Small, degraded and/or structured RNA via Agilent BioAnalyzer & Qubit RNA IQ Assay trna trna + % sirna Degraded RNA RIN#:. RIN#:. RIN#: N/A RNA IQ#:. Small RNA: % Large and/or Structured RNA: % RNA IQ#:. Small RNA: 9% Large and/or Structured RNA: % RNA IQ#:. Small RNA: % Large and/or Structured RNA: % Samples of trna, trna with % sirna in solution, or RNA exhaustively degraded by RNase were analyzed by Agilent Bioanalyzer and the Qubit RNA IQ assay on the Qubit Fluorometer.

14 Impact of highly structured RNA on small RNA samples % sirna; RIN =. % sirna; RIN =. % sirna; RIN =. % sirna; RIN =. RNA IQ# 9% sirna; RIN =. % sirna; RIN =. % % 9% % % % % %sirna in Yeast trna trna was combined with sirna in varying ratios and assayed, demonstrating the RNA IQ assay s sensitivity to different types of small RNA combined in the same sample.

15 qrt-pcr following RIN and IQ analysis of heat treated human liver samples T = minutes T = minutes T = minutes T = minutes Score 9 Minutes at C RIN # RNA IQ# Ct value hhif human sample hgapdh T = minutes RNA IQ#: 9. Agilent BioAnalyzer RIN#. Ct value Small RNA: % Large and/or Structured RNA: 9% human sample

16 qrt-pcr following RIN and IQ analysis of heat treated mouse liver samples T = minutes hhif T = minutes T = minutes Score 9 RIN # RNA IQ# Minutes at C Ct value mouse sample mgapdh NTC T = minutes Agilent BioAnalyzer RIN#. T = minutes RNA IQ#:. Ct value Small RNA: % Large and/or Structured RNA: % mouse sample NTC

17 qrt-pcr following RIN and IQ analysis of RNase treated human liver samples T = minutes NRF random primers 9 T = minutes T = minutes Quality Score Samples after RNase addition RNA IQ # RIN # Ct value control RNase treatment conditions GAPDH random primers T = minutes Agilent BioAnalyzer RIN#. RNA IQ#:. Small RNA: % Large and/or Structured RNA: % Ct value control RNase treatment conditions

18 qrt-pcr following RIN and IQ analysis of RNase treated human liver samples T = minutes NRF dt primers 9 T = minutes T = minutes Quality Score Samples after RNase addition RNA IQ # RIN # Ct value control RNase treatment conditions GAPDH dt primers T = minutes Agilent BioAnalyzer RIN#. RNA IQ#:. Small RNA: % Large and/or Structured RNA: % Ct value control RNase treatment conditions

19 NEW: Qubit RNA Integrity & Quality (IQ) Assay and Qubit Fluorometer Qubit & RNA IQ Assay Detect viable from degraded RNA with multiplexed dye assay: Unique dye for large and/or structured RNA Unique dye for small, degraded RNA Simple Add RNA sample to Qubit RNA IQ buffer Measure on Qubit Fluorometer Rapid time to result ~ minutes sample preparation ~ seconds per sample 9 For research use only. Not for use in diagnostic procedures Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.