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Focus: mRNA isolation for use in expression profiling

Isolating High-Quality mRNA From Total RNA with the PolyATtract® mRNA Isolation System For Use in Expression Profiling with the Pronto!™ Plus System

Poly(A)+ RNA was isolated from 293T and HeLa cells using the RNAgents® Total RNA Isolation System (Cat.# Z5110) in combination with the PolyATtract® mRNA Isolation System (Cat.# Z5200). Isolated poly(A)+ RNA was suitable for use with the Pronto!™ Plus System for microarray analysis.

By Patricia Fulmer, Ph.D., Dawn Rabbach, A.A.S., and Nadine Nassif, M.S.
Promega Corporation

Note: The data in this article were generated using the Pronto!™ Plus System. The ChipShot Labeling and Clean-Up System (Cat.# Z4000, Z4100), a component of the Pronto!™ Plus System, is now available separately for generating fluorescently labeled cDNA and should be used instead of the Pronto!™ Plus System in this protocol.

Introduction

The majority of researchers perform expression profiling experiments starting with total RNA. The Promega SV Total RNA Isolation System, a component of the Pronto!™ Plus Systems provides high-quality total RNA for expression profiling applications. Using 5µg of total RNA template, the Pronto!™ Plus System can detect as little as 3pg of target RNA.

For researchers who choose to use mRNA in expression profiling applications, Promega offers a variety of suitable isolation systems. The RNAgents® Total RNA Isolation System can be used to isolate total RNA on a larger scale than the SV Total RNA Isolation System, which is more appropriate for subsequent mRNA isolation. The RNAgents® System can be used to obtain total RNA from a number of sources, including animal and plant tissues and cultured cells. The PolyATtract® mRNA Isolation System is designed for rapid and convenient selection of mRNA from total RNA samples, including RNA purified with the RNAgents® System. Using total RNA as the starting material, the mRNA fraction can be isolated in approximately 45 minutes. In combination, these two systems provide an efficient method of isolating high-quality mRNA for expression profiling.

In these experiments we demonstrate the application of the RNAgents® and PolyATtract® Systems for use in expression profiling. cDNA synthesis and labeling, post-labeling clean-up, and subsequent hybridization to microarrays is accomplished with the Pronto!™ Plus System. The RNA isolated with the RNAgents® and PolyATtract® Systems serves as a suitable template for the cDNA labeling reaction in these procedures.

Isolation of mRNA From Total RNA

In this article we isolated mRNA from adherent cells for use with the Pronto™ Plus System. The RNAgents® Total RNA Isolation System Technical Bulletin (#TB087) contains protocols for the isolation of total RNA from other sources. Total RNA was isolated using a modification of the RNAgents® System protocol. Adherent cell samples were grown in T75 flasks, and prepared as described below. The rest of the procedure was performed as described in Technical Bulletin #TB087.

Sample Preparation

  1. Remove the media and wash the adherent cells with 20ml of 1X PBS. Make sure that the bottom of the flask is completely covered with PBS.
  2. Remove PBS and add 5ml of Invitrogen trypsin-EDTA solution (0.05% trypsin, 0.53mM EDTA•4Na; Cat.# 25300-054). Remember to completely cover the bottom of the flask. If the cells do not come off the bottom readily, place the flasks in a 37°C incubator until cells just begin to detach (1–2 minutes).
  3. Add 50ml of growth medium. Mix to drive the cells off the bottom.
  4. Transfer to a 50ml conical tube.
  5. Centrifuge at 1,400 × g for 5 minutes to pellet the cells. If the media is not clear, centrifuge for an additional 5 minutes.
  6. Remove media, and add 50ml of 1X PBS. Pipet to dissolve the pellet.
  7. Remove some of the cells and count with a hemacytometer.
  8. Centrifuge the cells at 1,400 × g for 5 minutes.
  9. Remove PBS, and add the appropriate volume of Denaturing Solution per Table 1 of Technical Bulletin #TB087.
  10. Continue with Section IV.C of Technical Bulletin #TB087.

The RNA yield was determined spectrophotometrically at 260nm, where 1 absorbance unit (A260)= 40µg of single-stranded RNA/ml. The purity of the RNA was estimated from the relative absorbances at 230, 260 and 280nm (i.e., A260/A280 and A260/A230). The total RNA yield and purity obtained with the RNAgents® System for cultured cells is shown in Table 1 and for mouse tissue in Table 2.

Table 1. Total RNA yield and purity from cultured cells using the RNAgents® Total RNA Isolation System.
Sample Average Number of Cells Average Yield (mg) Average A260/A230 Average A260/A280
293T (n=3) 1.1 × 108 3.2 ± 0.91 2.2 ± 0.01 1.7 ± 0.03
HeLa (n=3) 7.3 × 107 2.4 ± 0.31 2.1 ± 0.17 1.6 ± 0.09
Table 2. Total RNA yield and purity from mouse tissue using the RNAgents® Total RNA Isolation System.
Sample Amount of Tissue Processed Average Yield (mg) Average A260/A280
Intestine 1g 2.3 1.75
Spleen 1g 8.3 1.67
Lung 1g 1.9 1.75
Liver 1g 6.6 1.99
Kidney 1g 3.1 1.70
thumbnail-The quality and purity of total RNA isolated using the RNAgents® Total RNA Isolation System is confirmed using an Agilent 2100 Bioanalyzer.
The quality and purity of total RNA isolated using the RNAgents® Total RNA Isolation System is confirmed using an Agilent 2100 Bioanalyzer.

Figure 1. The quality and purity of total RNA isolated using the RNAgents® Total RNA Isolation System is confirmed using an Agilent 2100 Bioanalyzer. This image shows the integrity of total RNA isolated from cultured HeLa Cells. An rRNA ratio (28S/18S) of 2.4 was observed in this sample.

The integrity of the purified total RNA can be determined by denaturing agarose gel electrophoresis or by use of instrumentation such as an Agilent 2100 Bioanalyzer (Figure 1). The ratio of 28S to 18S eukaryotic ribosomal RNAs should be in the range of 1.5:1 to 2.5:1, indicating that no gross degradation of RNA has occurred.

We used the PolyATtract® mRNA Isolation System II (Cat.# Z5200) protocol for large-scale mRNA isolation (#TM021) to isolate mRNA from total RNA prepared with the RNAgents® System. Messenger RNA yield and purity were determined spectrophotometrically and are shown in Table 3.

Table 3. mRNA yield and purity using the PolyATtract® mRNA Isolation System.
Sample Average Amount of Input Total RNA (mg) Average mRNA Yield (µg) Average A260/A230 Average A260/A280
293T (n=3) 3.2 ± 0.91 22.1 ± 7.41 2.4 ± 0.08 2.1 ± 0.04
HeLa (n=3) 2.4 ± 0.31 12.1 ± 1.56 2.3 ± 0.08 2.0 ± 0.13
thumbnail-Cy®3- and Cy®5-labeled cDNA hybridized to a DNA microarray.
Cy®3- and Cy®5-labeled cDNA hybridized to a DNA microarray.

Figure 2. Cy®3- and Cy®5-labeled cDNA hybridized to a DNA microarray. Cy®3-labeled cDNA from 293T mRNA and Cy®5-labeled cDNA from HeLa mRNA were hybridized to custom 4K arrays using the Pronto!™ Plus System and data were acquired using a Genepix® 4000B scanner (Axon Instruments, Inc.). A representative subgrid is shown.

Microarray Analysis

The purified mRNA was used as a template to generate fluorescently labeled cDNA. The Pronto!™ Plus System was used to generate the cDNA via direct labeling with 1.5µg of mRNA template per reaction. The cDNA yield and the frequency of incorporation (FOI) are shown in Table 4. The Cy®3- and Cy®5-labeled cDNA were hybridized to a DNA microarray (Corning) as shown in Figure 2. Messenger RNA isolated from 293T cells was used as a template for Cy®3-labeled cDNA, while mRNA isolated from HeLa cells was used as a template to prepare Cy®5-labeled cDNA. Hybridization was performed using the Pronto!™ Plus System. For hybridization, 4pmol of each of the labeled cDNAs was mixed together and applied to arrays printed on UltraGAPS™ slides under 22 × 22mm coverslips.

Table 4. Yield of Fluorescently Labeled cDNA using the Pronto!™ Plus System.
Label Yield (ng) pmol Dye Incorporated FOI
Cy®3 (n=8) 446 ± 22 57 ± 5 42 ± 2
Cy®5 (n=8) 452 ± 18 32 ± 2 23 ± 1

To demonstrate reproducibility the average Relative Fluorescent Units (RFU) for spot signal and background were compared (Figure 3). We observed high levels of reproducibility and signal-to-background in these experiments.

thumbnail-Signal and background intensity of microarray hybridizations using either mRNA template purified from RNAgents®-PolyATtract® Systems or from commercial mRNA.
The quality and purity of total RNA isolated using the RNAgents® Total RNA Isolation System is confirmed using an Agilent 2100 Bioanalyzer.

Figure 3. Signal and background intensity of microarray hybridizations using either mRNA template purified from RNAgents®-PolyATtract® Systems or from commercial mRNA. The RNAgent®-PolyATract® Systems were used in combination to isolate mRNA from 8 HeLa and 293T cell culture samples as described. The ChipShot™ Labeling System was used to convert 1.5µg of the purified mRNA from 293T and HeLa cells into Cy®3-labeled and Cy®5-labeled cDNA, respectively. Four picomoles of each labeled cDNA were combined for complex hybridizations to custom 4K arrays produced by Corning Life Sciences following the Pronto!™ Universal protocol. Median signal and background for the Cy®5 channel and the Cy®3 channel were plotted (blue bars, with standard deviation), and compared to results using commercially-acquired RNA as starting material (burgundy bars). No significant variation is seen among RNAgent®-PolyATract®-derived hybridizations compared to commercially-derived hybridizations.

Conclusions

The PolyATtract® System is a quick, convenient method of isolating pure, intact mRNA. Messenger RNA purified from RNAgents® purified total RNA using the PolyATtract® mRNA Isolation System performs well as a template for generating fluorescently labeled cDNAs with the Pronto!™ Plus System. Downstream hybridizations to microarrays were reproducible and demonstrated good signal-to-background ratios.

Acknowledgments

We would like to acknowledge Corning Incorporated for substantial contributions in the co-development of the Pronto!™ Plus Systems and Agilent Technologies for providing the 2100 Bioanalyzer.

Addendum

In June 2005, two new Pronto!™ Plus Systems were launched jointly by Promega Corporation and Corning Incorporated. The Pronto!™ Plus Direct System provides reagents and protocols for the synthesis and hybridization of fluorescently-labeled cDNA generated via direct incorporation of Cy®-labeled nucleotides. The Pronto!™ Plus Indirect System labels cDNA by incorporating aminoallyl-modified nucleotides during cDNA synthesis, and then conjugating an NHS-ester dye to the aminoallyl-modified cDNA after the reverse transcription reaction is complete. Both new Pronto!™ Plus Systems include the new ChipShot™ Membrane Clean-Up System for purification of Cy®-labeled cDNA.

The labeled cDNA used to generate hybridization data in the above article was synthesized with the original Pronto!™ Plus System, which used a particle-based cDNA purification method. The ChipShot™ Membrane Clean-Up System included with the new Pronto!™ Plus Systems provides a number of advantages over the previous purification method, including ease-of-use and improved cDNA recovery. Tables 5 and 6 indicate the cDNA yield and labeling efficiency achieved using 1.5µg of mRNA template per reaction in the new Pronto!™ Plus Systems. Hybridization results are comparable when using the new ChipShot™ Membrane Clean-Up System (1) compared to the previous particle-based cDNA purification method.

Table 5. Yield of Fluorescently Labeled cDNA using the Pronto!™ Plus Direct System.
Label Yield (ng) pmol Dye Incorporated FOI
293T mRNA
Cy®3 (n=3) 350 ± 30 60 ± 5 56 ± 0
Cy®5 (n=3) 412 ± 8 46 ± 1 36 ± 0
HeLa mRNA
Cy®3 (n=3) 413 ± 11 70 ± 2 55 ± 0
Cy®5 (n=3) 422 ± 12 48 ± 1 37 ± 2
Table 6. Yield of Fluorescently Labeled cDNA using the Pronto!™ Plus Indirect System.
Label Yield (ng) pmol Dye Incorporated FOI
293T mRNA
Cy®3 (n=3) 1322 ± 106 92 ± 6 23 ± 2
Cy®5 (n=3) 1215 ± 93 63 ± 9 17 ± 1
HeLa mRNA
Cy®3 (n=3) 1331 ± 92 95 ± 3 23 ± 1
Cy®5 (n=3) 1386 ± 121 89 ± 18 21 ± 3

Reference

  1. Canel, C., Bunch, T.A. and Nassif, N. (2005) Pronto!™ Plus Systems: New Integrated Reagent Systems for Optimal Microarray Performance. Promega Notes 90, 5–9.