Promega Corporation

Gene Expression: RNA Analysis Workflow

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Abstract

Whether your downstream application is Northern blotting, microarray analysis, RNA-Seq or RT-qPCR, every step of the RNA analysis process is important. In this article we describe and evaluate products for every step of the RNA analysis workflow for both cultured cells and formalin-fixed, paraffin-embedded tissue (FFPE). Products evaluated included: the Maxwell® 16 LEV simplyRNA Cells Kit, ReliaPrep™ RNA Cell Miniprep System and ReliaPrep™ FFPE Total RNA Miniprep System (purification);  RNasin® Ribonuclease Inhibitor and QuantiFluor™ RNA System (protection and quantification, respectively); and GoTaq® 1-Step and 2-Step RT-qPCR Systems (amplification).

Doug Wieczorek, Laurence Delaurière and Trista Schagat

Promega Corporation Publication Date: tpub100; February 2013

Introduction

RNA analysis and related techniques are used to monitor gene expression, study genetic regulation and determine disease biomarkers. Whether the desired downstream application is Northern blotting, microarray analysis, RNA-Seq or RT-qPCR, consideration must be given to each step of the RNA analysis workflow, from purification to protection, quantitation and amplification. Here we evaluate workflow solutions for RNA analysis of cultured cells and formalin-fixed, paraffin-embedded (FFPE) tissue. For purification, the Maxwell® 16 LEV simplyRNA Cells Kit, ReliaPrep™ RNA Cell Miniprep System and ReliaPrep™ FFPE Total RNA Miniprep System were evaluated. For protection and quantitation, RNasin® Ribonuclease Inhibitor and the QuantiFluor™ RNA System were used, respectively. For amplification, the GoTaq® 1-Step and 2-Step RT-qPCR Systems were compared. Together, these products offer a complete, compatible solution for RNA analysis (Figure 1).

11160TA_1015pxFigure 1. Complete solution for RNA analysis.

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Purify

There are a number of key challenges when performing RNA purifications. With any RNA sample it is critical to maintain and maximize RNA integrity. Ideal purification methods include efficient RNase inactivation and simple procedures that minimize hands-on time and the possibility of introducing RNases. Purity is also a major consideration. RNA contaminated with residual purification reagents (e.g., alcohols or salts) or genomic DNA may result in under- or overestimation of specific target RNAs. When samples are limiting, it is also important to be able to purify RNA at high concentration to maximize the likelihood of success in downstream assays. We purified RNA from tissue culture cells or FFPE mouse liver tissue sections using three RNA purification systems, the ReliaPrep™ RNA Cell Miniprep System, ReliaPrep™ Total RNA FFPE Miniprep System and Maxwell® LEV simplyRNA Purification Kits. Each of these includes an in-process DNase treatment and minimizes the hands-on time required when compared to existing methods.

Maxwell® 16 LEV simplyRNA Cells Kit

The Maxwell® 16 LEV simplyRNA Cells Kit is designed for automated RNA purification from fresh or frozen cells on the Maxwell® 16 Instrument (1) . The procedure uses a DNase step and a low elution volume (LEV) format that combine to isolate concentrated high-quality RNA suitable for use in many downstream applications such as quantitative RT-PCR and RNA-Seq. We isolated RNA in triplicate from 1 million HEK293 cells using the Maxwell® 16 LEV simplyRNA Cells Kit according to the Maxwell® 16 LEV simplyRNA Cells Kit Technical Manual #TM351, and the purified RNA was eluted in 50µl of Nuclease-Free Water.

ReliaPrep RNA Cell Miniprep System

The ReliaPrep™ RNA Cell Miniprep System provides a fast and simple manual method for purifying intact total RNA from as few as 100 cultured cells and takes as little as 30 minutes. The system incorporates a DNase treatment step directly on the minicolumn membrane, and RNA can be eluted in as little as 15µl, resulting in pure RNA that does not require additional purification or concentration for use in demanding applications. We isolated RNA in triplicate from 1 million HEK293 cells using the ReliaPrep™ RNA Cell Miniprep System according to ReliaPrepRNA Cell Miniprep System Technical Manual #TM370. For the experiments described here, the purified RNA was eluted in 30µl of Nuclease-Free Water.

ReliaPrep FFPE Total RNA Miniprep System

The ReliaPrep™ FFPE Total RNA Miniprep System provides a complete, all-inclusive method for purification of quality total RNA from formalin-fixed, paraffin-embedded tissue without using hazardous solvents or overnight digestion. Total RNA can be isolated from FFPE tissue in approximately two and one-half hours with minimal hands-on time.

The ReliaPrep™ FFPE Total RNA Miniprep System provides a complete method for purifying quality total RNA from FFPE tissue. The deparaffinization step occurs without harsh organic solvents or an overnight digestion. Total RNA can be isolated from FFPE tissue in approximately two and one-half hours including the DNase treatment and with minimal hands-on time. The resulting RNA is intact, amplifiable total RNA. We isolated RNA in triplicate from FFPE mouse liver tissue sections using the ReliaPrep™ FFPE Total RNA Miniprep System according to the ReliaPrepFFPE Total RNA Miniprep System Technical Manual #TM353, and the purified RNA was eluted in 50µl of Nuclease-Free Water.

Protect

High-integrity RNA is required for many downstream applications. However, RNA is vulnerable to the effects of RNases in the environment, which can lead to RNA degradation and potentially the total loss of the RNA sample. When working with RNA, every effort should be made to prevent the introduction of these RNases into the laboratory, but complete removal can be difficult no matter what steps are taken. Following purification, ribonuclease inhibitors can be used to protect RNA from RNases that may be introduced during subsequent handling.

RNasin® Ribonuclease Inhibitors

RNasin® Ribonuclease Inhibitors have been cited as the source of RNase protection in over 9,000 papers, making RNasin® Ribonuclease Inhibitors the most trusted reagent for RNA protection. RNasin® Ribonuclease Inhibitor is a protein that specifically binds and inactivates RNases. RNasin® Ribonuclease Inhibitors have broad-spectrum RNase inhibitory properties and inhibit common eukaryotic RNases including RNase A, RNase B and RNase C. RNasin® Ribonuclease Inhibitors are useful for the long-term protection of any RNA stocks (2) or in any application where eukaryotic RNase contamination is a potential problem, such as in vitro transcription/translation and RT-PCR (3) .

Quantitate

Proper care taken during RNA purification, handling and storage can mean the difference between success and failure in downstream applications. Selecting and consistently using an appropriate quantitation method may be just as important. There are a number of options for RNA quantitation, each having specific advantages and disadvantages (4) . Some methods only provide information on RNA concentration, while others may provide insight into the purity and integrity of the RNA sample. For greatest sensitivity, specificity and ease-of-use, we quantitated RNA purified from tissue culture cells and FFPE mouse liver tissue sections using the QuantiFluor™ RNA System fluorescent dye-based approach. Results were compared to UV absorbance and the Agilent 2100 Bioanalyzer (Table 1 and Figure 2).

11204LATable 1. The Concentration and Purity of RNA Purified from Cells and FFPE Tissue as Determined by Various Quantitation Methods

RNA purity ratios were calculated by UV absorbance, RNA yield was calculated based on recovered elution volume and RNA concentration was measured by the QuantiFluor™ RNA System. The average of triplicate purifications is shown. The standard deviations are shown in parentheses.

RNA purity ratios were calculated by UV absorbance, RNA yield was calculated based on recovered elution volume and RNA concentration was measured by the QuantiFluor™ RNA System. The average of triplicate purifications is shown. The standard deviations are shown in parentheses.

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11170TA_965pxFigure 2. Electrophoretic traces and RIN values of RNA purified from cells and FFPE tissue using the 2100 Bioanalyzer instrument.

The 18S and 28S rRNA peaks are prominent in RNA from cells. N/A: Samples with RIN values that were unable to be determined.

The 18S and 28S rRNA peaks are prominent in RNA from cells. N/A: Samples with RIN values that were unable to be determined.

/~/media/images/resources/figures/11100-11199/11170ta_965px.jpg?la=en

QuantiFluor RNA System

The QuantiFluor™ RNA System contains a fluorescent RNA-binding dye that enables sensitive quantitation of small amounts of RNA in solution. The system significantly increases sensitivity over absorbance at 260nm for samples that are low in concentration, and since less template is required than traditional spectrophotometry, precious samples can be saved for more important downstream assays. This system can be used on any fluorescent instrument with appropriate optical channels, including the QuantiFluor™ Single-Tube Fluorometers and the GloMax®-Multi+ Detection System. The RNA purified from cells and FFPE tissue was quantitated using the QuantiFluor™ RNA System using the QuantiFluor™-ST Instrument (5) . The results are shown in Table 1.

The RNA purified from cells using the Maxwell® 16 LEV simplyRNA Cells Kit and the ReliaPrep™ RNA Cell Miniprep System was of high concentration. All samples had A260/A280 and A260/A230 purity values >2.0 and RIN values >8.5, indicative of very high quality RNA. For RNA purified from FFPE mouse liver tissue, while A260/A280 values were also >2.0, A260/A230 values averaged 1.17 and RIN values were unable to be determined due to low integrity. This is typical for RNA purified from FFPE tissue as the fixation process, sample storage and overall sample quality can significantly affect the overall quality of the resulting RNA, leading to reduced purity and integrity. Differences in the apparent RNA concentrations using the three different methods are a reflection of the different means by which each method determines RNA concentration. It is important to consistently use the same quantitation method if comparisons need to be made for further downstream analysis.

Amplify

Reverse transcription-quantitative PCR (RT-qPCR) can be used to quantitate specific target RNA using primers specific to the RNA of interest. Under optimal reaction conditions, a single copy of a target sequence can be detected making RT-qPCR an extremely sensitive method of detection. With low copy or rare transcripts, a more sensitive assay might mean the difference between detecting a rare transcript or missing it completely. We analyzed RNA purified from tissue culture cells and FFPE mouse liver tissue sections by one-step and two-step RT-qPCR using the GoTaq® 1-Step and 2-Step RT-qPCR Systems. The GoTaq® 1-Step and 2-Step RT-qPCR Systems include RNasin® Ribonuclease Inhibitor in the reaction mixes for improved stability of the RNA template during cDNA synthesis.

GoTaq® 1-Step RT-qPCR System

The GoTaq® 1-Step RT-qPCR System is a system for quantitative analysis of RNA using a one-step RT-qPCR protocol in a single tube. The use of BRYT Green® Fluorescent Dye in GoTaq® Real-Time PCR Systems allows a brighter signal compared with the traditional SYBR® Green dye. When used in combination with GoScript™ Reverse Transcriptase, it results in sensitive detection with the convenience of a single reaction setup. For RNA purified from cell culture cells, 25ng of RNA was reverse transcribed and the cDNA amplified in a 50µl reaction volume using human HPRT1 primers. The data was analyzed on the CFX96™ Real-Time PCR Detection System (Bio-Rad). For RNA purified from FFPE mouse liver tissue, 25ng of RNA was reverse transcribed and the cDNA amplified in a 50µl reaction volume using mouse beta-2-microglobulin primers. The data was analyzed on the 7500 Real-Time PCR System (Applied Biosystems). The results are shown in Figure 3.

GoTaq® 2-Step RT-qPCR System

The GoTaq® 2-Step RT-qPCR System is a system for quantitative analysis of RNA using a two-step RT-qPCR protocol. The system uses the same BRYT Green® Fluorescent Dye and GoScript™ Reverse Transcriptase as the GoTaq® 1-Step RT-qPCR System for efficient reverse transcription and sensitive detection of rare and abundant transcripts. For RNA purified from cells, 50ng of RNA was reverse transcribed using random primers according to the GoTaq® 2-Step RT-qPCR System Technical Manual #TM337, and 5µl of cDNA (25% of the total) was then amplified in a 50µl reaction volume using human HPRT1 primers. The data was analyzed on the CFX96™ Real-Time PCR Detection System (Bio-Rad). For RNA purified from FFPE mouse liver tissue, 50ng of RNA was reverse transcribed using random primers according to the GoTaq® 2-Step RT-qPCR System Technical Manual #TM337, and 10µl of cDNA (50% of the total) was then amplified in a 50µl reaction volume using mouse beta-2-microglobulin primers. The data was analyzed on the 7500 Real-Time PCR System (Applied Biosystems). The results are shown in Figure 3.

11188MA_485pxFigure 3. Real-time PCR analysis of RNA purified from cells and FFPE tissue using the GoTaq® 1-Step and 2-Step RT-qPCR Systems.

Input RNA for each reaction was based on the QuantiFluor™ RNA System. The average Cq of triplicate purifications with standard deviations are shown.

Input RNA for each reaction was based on the QuantiFluor™ RNA System. The average Cq of triplicate purifications with standard deviations are shown.

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HPRT1 RNA (human cells) and beta-2-microglobulin RNA (FFPE mouse liver tissue) was successfully detected following reverse transcription and amplification using the GoTaq® 1-Step and 2-Step RT-qPCR Systems. The 1-step procedure amplified 1–4 Cq later than the 2-step procedure, suggesting the best sensitivity may be gained by a 2-step reaction. The RNA purified from cells was of high purity and integrity and expected to perform well in any downstream application tested. However, the RNA purified from FFPE mouse liver tissue was of lower purity and highly fragmented due to the inherent nature of FFPE samples as indicated by UV absorbance and Bioanalyzer analysis. However, the overall quality of the RNA purified using the ReliaPrep™ FFPE Total RNA Miniprep System in combination with the sensitivity and efficiency of the GoTaq® RT-qPCR Systems and the added protection of RNasin® Ribonuclease Inhibitor resulted in the successful detection of the beta-2-microglobulin target RNA.

Summary

The quality of data achieved from RNA expression analysis experiments depends on thoughtful execution of each step in the RNA workflow. For the best results, choose a complete workflow solution that offers high-quality, easy-to-use and reliable products. Using the Maxwell® 16 simplyRNA Kits or ReliaPrep™ RNA Miniprep Systems, you will have purified RNA that is intact and free of contaminants that may confound downstream results. Because RNA is vulnerable to the effects of RNases in the environment, which can lead to RNA degradation and potentially the total loss of the RNA sample, it needs to be protected with RNasin® Ribonuclease Inhibitor to maintain integrity throughout subsequent analyses. Selecting and consistently using an appropriate quantitation method is important. Some methods only provide information on RNA concentration, while others may provide insight into the purity and integrity of the RNA sample. We found the QuantiFluor™ RNA System to be a sensitive and specific approach. Be careful not to over-interpret quality assessment measurements by absorbance or Bioanalyzer as they do not always predict downstream performance. Finally, the amplification method you use needs to be robust and sensitive. Reverse transcription-quantitative PCR (RT-qPCR) can quantitate specific target RNA using primers specific to the RNA of interest, and under optimal reaction conditions, a single copy of a target sequence can be detected. Here we used the GoTaq® 1-Step and 2-Step RT-qPCR Systems with BRYT Green® Dye for sensitive earlier detection of our targets.

References

  1. Gorshe, R. and Wieczorek, D. (2011) Maxwell® 16 LEV simplyRNA Cell and Tissue Kits: A comparison to QIAcube® and TRIzol®. Promega Corporation Website.
  2. Bratz, M., Hook, B. and Schagat, T. (2012) Making sense of your genetic reporter data through assay multiplexing. PubHub Online.
  3. Hooper, K. (2004) RNasin® Ribonuclease Inhibitors: Superior performance for all of your RNA analysis needs. Promega Notes 86, 25–7.
  4. Wieczorek, D. et al. (2013) Methods of RNA quality assessment. Submitted.
  5. (2012) Measuring the QuantiFluor™ RNA System Using the QuantiFluor™-ST Fluorometer Applications Note. Promega Corporation Website.

How to Cite This Article

Wieczorek, D., Delaurière, L. and Schagat, T. Gene Expression RNA Analysis Workflow. [Internet] tpub100; February 2013. [cited: year, month, date]. Available from: http://www.promega.co.uk/resources/pubhub/tpub_100-gene-expression-rna-analysis-workflow/

Wieczorek, D., Delaurière, L. and Schagat, T. Gene Expression RNA Analysis Workflow. Promega Corporation Web site. http://www.promega.co.uk/resources/pubhub/tpub_100-gene-expression-rna-analysis-workflow/ Updated tpub100; February 2013. Accessed Month Day, Year.

Figures

11160TA_1015pxFigure 1. Complete solution for RNA analysis.

/~/media/images/resources/figures/11100-11199/11160ta_1015px.jpg?la=en
11170TA_965pxFigure 2. Electrophoretic traces and RIN values of RNA purified from cells and FFPE tissue using the 2100 Bioanalyzer instrument.

The 18S and 28S rRNA peaks are prominent in RNA from cells. N/A: Samples with RIN values that were unable to be determined.

The 18S and 28S rRNA peaks are prominent in RNA from cells. N/A: Samples with RIN values that were unable to be determined.

/~/media/images/resources/figures/11100-11199/11170ta_965px.jpg?la=en
11188MA_485pxFigure 3. Real-time PCR analysis of RNA purified from cells and FFPE tissue using the GoTaq® 1-Step and 2-Step RT-qPCR Systems.

Input RNA for each reaction was based on the QuantiFluor™ RNA System. The average Cq of triplicate purifications with standard deviations are shown.

Input RNA for each reaction was based on the QuantiFluor™ RNA System. The average Cq of triplicate purifications with standard deviations are shown.

/~/media/images/resources/figures/11100-11199/11188ma_485px.jpg?la=en

Tables

11204LATable 1. The Concentration and Purity of RNA Purified from Cells and FFPE Tissue as Determined by Various Quantitation Methods

RNA purity ratios were calculated by UV absorbance, RNA yield was calculated based on recovered elution volume and RNA concentration was measured by the QuantiFluor™ RNA System. The average of triplicate purifications is shown. The standard deviations are shown in parentheses.

RNA purity ratios were calculated by UV absorbance, RNA yield was calculated based on recovered elution volume and RNA concentration was measured by the QuantiFluor™ RNA System. The average of triplicate purifications is shown. The standard deviations are shown in parentheses.

/~/media/images/resources/tables/11200-11299/11204la.jpg?la=en

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