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Focus: RNA Isolation from Plants

Titration of Tomato Leaf Material Used as Starting Material for SV Total RNA Isolation

By Hemanth Shenoi, Ph.D., and Daniel Kephart, Ph.D.
Promega Corporation

Introduction

This article details experiments aimed at demonstrating how much plant material (tomato leaf) can be used for total RNA isolation using the SV Total RNA Isolation System^(a) and also to determine the linear range of yield with this system. The isolated total RNA was analyzed by spectrophotometry and reverse transcriptase-PCR (RT-PCR)^(b).

Experimental Conditions

RNA Extraction: Frozen tomato leaf tissue (940mg) was ground in liquid nitrogen using a mortar and pestle. Initially, 500mg of tissue was solubilized in 1.3ml SV RNA Lysis Buffer. To this lysate, SV RNA Dilution Buffer (2.6ml) was added. The samples were centrifuged for 10 minutes at 14,000 x g, and ethanol (1.2ml) was added to the supernatant. Volumes of lysate corresponding to 30, 100, 200 and 500mg of original tissue mass were passed through the SV RNA Spin Basket membrane and processed as recommended (2, SV Total RNA Isolation System Technical Manual #TM048). Note that multiple spins were required to pass the 200mg and 500mg samples through a single Spin Basket membrane; no clogging of the Spin Basket membrane was observed.

RT-PCR Amplification: RNA was next analyzed by RT-PCR using primer pairs specific for the RUBISCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) small subunit gene (rbcS3A) mRNA, a common and abundant plant RNA. Oligonucleotide primers were chosen within highly conserved sequences of the rbcS3A gene. The primer sequences used were:

Primer Sequences

Upstream
5´-CCCTTCACTGGACTCAAGTCCACCGC-3'

Downstream
5´-GCTTGTAAGCGATGAAGCTGATGCACTGC-3'

Each total RNA sample (1ľl from a total of 100ľl) was reverse transcribed and amplified using Promega's Access RT-PCR System^(b) (Cat.# A1250, A1280). The cycling profile was as follows:

First Strand cDNA Synthesis
1 cycle	48°C for 45 minutes

1 cycle	94°C for 2 minutes

Second Strand cDNA Synthesis and PCR Amplification
35 cycles	94°C for 30 seconds 60°C for 1 minute 68°C for 1 minute

1 cycle	68°C for 7 minutes

1 cycle	4°C soak

After amplification, the products (10ľl) were analyzed on a 1.5% agarose gel (Figure 1).

Results

The purity of the tomato leaf RNA isolated with the SV Total RNA Isolation System was high, as measured by UV absorbance ratios. Pure preparations of RNA have an A₂₆₀/A₂₈₀ ratio of approximately 2.0. If there is protein contamination in the sample, this ratio will be significantly lower (1). A₂₆₀/A₂₃₀ ratios lower than 1.5 generally indicate carryover contamination by guanidine thiocyanate. The A₂₆₀/A₂₃₀ ratios for the tomato leaf RNA samples isolated with this system were greater than or equal to 2.2, and along with the A₂₆₀/A₂₈₀ ratios, indicate high purity of the RNA (Table 1).

Table 1. Yield and Purity of Total RNA from Tomato Leaves.

Amount of Tissue Sample (mg)	Yield per Prep (ľg)	Yield per mg of Tissue (ľg)	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀
30	5.7	0.19	1.7	2.2
100	16.6	0.16	2.0	2.2
200	33.0	0.17	2.2	2.2
500	92.0	0.18	2.3	2.3

Each value represents the average of duplicate spectrophotometer readings of a single sample.

RNA was isolated from tomato leaf, yielding RNA that was free of detectable genomic DNA in PCR analysis (without reverse transcriptase added) (Figure 1, lane 5). The negative control amplification, which contained RNA from 500mg of tissue without AMV Reverse Transcriptase added, resulted in no product (showing no detectable contaminating genomic DNA.

Figure 1. RNA isolated from titrated amounts of starting tomato leaf and used as template in RT-PCR. Aliquots (1ľl of 100ľl) of the RNA isolated from the indicated tissue masses were used as template in RT-PCR. After amplification, 10ľl (one-fifth) of each reaction were separated by agarose gel electrophoresis. Lane 1, 30mg tomato leaf; lane 2, 100mg tomato leaf; lane 3, 200mg tomato leaf; lane 4, 500mg tomato leaf; and lane 5, 500mg tomato leaf without reverse transcriptase in the RT-PCR reaction. Lane M, Promega’s 100bp DNA Ladder (Cat.# G2101); bands in lane M correspond to 500, 400, 300 and 200bp. Primers for RT-PCR are to the ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO) gene.

Summary

The SV Total RNA Isolation System performed well with up to 500mg of tomato leaf material per preparation. The amount of nucleic acid isolated from a sample was linear over the range tested (30mg to 500mg), and spectrophotometric analysis indicated that the RNA is of high quality. All of the samples performed well in RT-PCR amplifications using the RUBISCO primer pair, and there was no detectable genomic DNA in the preparations.

References

Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Laboratory, Cold Spring Harbor, NY.
SV Total RNA Isolation System Technical Manual #TM048.

^(a)Patent Pending.

^(b)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Use of this product is recommended for persons that either have a license to perform PCR or are not required to obtain a license.

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