Genes exist in cells as DNA and are expressed by first being transcribed into an RNA copy. Actively expressed genes may be represented by thousands of RNA copies in a cell at any given time. In tissue exposed to a given treatment, the level of expression of several genes may change significantly due to changes in RNA synthesis or degradation, possibly altering the total amount of RNA in the tissue. One method used in studies to determine the change in the expression level of a gene of interest is known as quantitative real-time PCR (qRT-PCR). This method relies on the polymerase chain reaction (PCR), a technique which results in the multiplication of a specific DNA sequence; samples with more copies of the gene result in more rapid amplification of that gene. The specificity of the process is determined by the use of "primers," short pieces of DNA matching the gene of interest. The qRT- PCR process begins with converting the RNA back into DNA copies, known as cDNA (complementary DNA) using a naturally-occurring enzyme that performs this function, then amplifying the cDNA. The equipment used to carry out qRT- PCR determines the number of copies as they are formed, resulting in accurate comparisons of the original copy numbers of the gene in the samples. Typically, this method is used to estimate the number of copies of a transcript of interest in an aliquot of total RNA extracted from tissue samples before and after treatment. This determination is influenced by the amount of total RNA in the tissue. For example, if total RNA increased 2-fold, and the occurrence of the target transcript increased 10-fold, estimating the number of transcript copies in an aliquot of RNA would indicate only a five-fold increase in the target transcript, while a 10-fold increase had actually occurred in each cell. Here, we suggest a simple method using qRT-PCR to estimate the relative proportions of total RNA and a specific RNA transcript in tissue samples.

Materials and Methods

Plant culture

Wheat (Triticum aestivum L.) line 442, a winter wheat germplasm and line 443, a spring wheat germplasm (Storlie et al. 1998) were used. Plants were grown under 16h light at 20ºC for 14d, then were transferred to a chamber with the same light conditions at 2ºC. The second leaves (of three) to open on the plants were collected at the time of transfer and after 7d growth at 2ºC. To determine the amount of water in the plant samples, second leaves from each of the treatments were weighed, dried for 24 hrs at 80ºC, and then weighed again. Water content was determined by subtraction. All moisture determinations and PCR analyses were replicated three times using independent plants.

RNA extraction

Three types of samples were processed; plant tissue from each temperature treatment alone and a sample consisting of equal amounts of plant tissue from the two treatments. The same mass of tissue from each plant was pulverized in a mortar and pestle in liquid nitrogen. Trizol reagent (Invitrogen, San Diego, CA) was added to the mortar and total RNA was extracted according to the manufacturers directions. The final RNA pellet was dissolved in DEPC-treated water and quantified with UV spectrophotometry.

qRT- PCR

Quantitative real-time PCR was carried out on a RotorGene 2000 (Corbett Research, Sydney, Australia) using a SYBR green detection protocol (Karsai et al. 2002). The primers used were specific for genes encoding wheat manganese-superoxide dismutase and phospholipase D. QRT-RT PCR was carried out as described by Baek and Skinner, 2003.

The template for qRT-PCR comprised six treatments consisting of mixtures of RNA from the two treatments. A seventh treatment consisted of RNA extracted from combined equal amounts of control and cold-treated plant tissue.

Calculation of RNA content

The qRT-PCR process is used to determine the average number of copies of the gene under study in the treated tissue, the control tissue, and the combined tissue sample. From this information, two equations are written: Tt + Cc = m and T + C = N where, in the mixed tissue sample, N = the amount of total RNA analyzed from the mixed tissue sample, T = the amount of total RNA from the treated tissue, C = the amount of total RNA from the control tissue, and m = the average number of copies of the gene in the mixed tissue sample. From the treated tissue and the control tissue analyzed separately, t = the average number of copies of the gene in the treated tissue, and c = the average number of copies of the gene in the control tissue. T and C are unknown initially, but these two equations can be solved simultaneously, revealing T and C. From simply dividing T/C, the change in the amount of total RNA due to the treatment is determined.

Concluding Remarks

Using this qRT-PCR method, we calculated an average increase of total RNA, including the spring and winter wheat lines, of 2.3-fold after seven days of exposure to cold temperature. Sarhan and D’Aoust, 1975, using a method of extraction and quantification of nucleic acids, first measured RNA change after 11 days of cold acclimation and estimated an average increase of total RNA, including spring and winter wheat, of 2.1-fold. Our finding of an average 2.3-fold increase in total RNA and the 2.1 fold increase found by Sarhan and D’Aoust, 1975 were within one standard deviation unit of our data, and therefore were not considered significantly different, suggesting the qRT-PCR method we propose here provides a rapid and reliable method of estimating changes in total RNA content in response to treatments. This information may then be used to calculate the actual change in the number of copies of the RNA transcript in each unit mass of tissue.

Although we worked with plant tissue, the method we have described here is not in any way limited to plant studies. In addition to the study in wheat (Sarhan and D’Aoust, 1975), there have been several reports of total RNA increase or decrease in response to, for example, disease in rats (Kent et al. 1991), growing conditions in a nematode (Fabian and Johnson, 1995), and nutrition status in fish (Gwak and Tanaka, 2001). Significant variation of total RNA amounts in different tissues of sea urchin also have been reported (Phillips, 1982). The method we have described here can easily be applied to biological samples of any kind, and should provide information on changes in total RNA content in response to any treatment of interest, or in different tissues.

References

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