Quantitative RT -PCR of RcSCR2

From the results of the RcSCR2 RT-PCR it was not possible to quantify the RcSCR2 mRNA in the endosperm. It was difficult to generate a reliable result by a simple PCR. The more reliable method is real time RT-PCR.

In an attempt to investigate the RcSCR2 expression level in the endosperm, the total RNA from different day old endosperm was isolated. Afterwards the total RNA was treated with RNase free DNase I to avoid contamination by genomic DNA.

To obtain an internal control to monitor the reverse transcription and subsequent PCR amplification, an artificial control was added to the total RNA sample from the endosperm.

The in vitro transcription product of nsLTPc1 was used as the artificial control. The advantage is that nsLTPc1 is not expressed in the endosperm. 10 ng of the nsLTPc1 transcript were added to each total RNA sample. Subsequently the reverse transcription was performed using the oligo-(dT)15 primer. In order to obtain a reliable result, different primers were designed by program analysis. These primers were tested by RT-PCR and the products were checked in an agarose gel. The SCR2RTF1 primer and SCR2RTR1 primer were used for Real time RT-PCR, because no nonspecific amplification product was found in the agarose gel (Figure 45).

Different amounts of these two primers were used for optimization of the PCR. The result was checked in a 2% agarose gel. The best combination was 15 pmole for both primers in a 50 µl PCR reaction. Afterward s different c ycles of PCR were performed. No amplification products could be found when the PCR cycle number was less than 24. But 28, 32, 36 and 40 cycles of amplification could amplify sufficient PCR product (Figure 47).

Table 14: The primes for real time RT-PCR for RcSCR2.

Primer Sequence

Figure 45 : RT-PCR of RcSCR2 and nsLTPc1. Total RNA of the endosperm was used as a template. M:

100 bp DNA marker. 1-5: RcSCR2 RT-PCR products. 6-10: nsLTPc1 RT-PCR products. Lane 1 and 6:

day 2; Lane 2 and 7: day 3; Lane 3 and 8: day 4; Lane 4 and 9: day 5; Lane 5 and 10: day 6.

Figure 46 : The optimization of primer concentrations for real time RT-PCR of RcSCR2. M:

pUC19/MscI marker. Lane 1:SCR2RTF1: 4 pmole; SCR2RTR1: 4 pmole. Lane 2: SCR2RTF1: 4 pmo le;

SCR2RTR1: 15 pmole. Lane 3: SCR2RTF1: 4 pmole; SCR2RTR1: 45 pmole. Lane 4: SCR2RTF1: 15 pmole; SCR2RTR1: 4 pmole. Lane 5: SCR2RTF1: 15 pmole; SCR2RTR1: 15 pmole. Lane 6: SCR2RTF1:

15 pmole; SCR2RTR1: 45 pmole. Lane 7: SCR2RTF1: 45 pmole; SCR2RTR1: 4 pmole. Lane 8:

SCR2RTF1: 45 pmole; SCR2RTR1: 15 pmole. Lane 9: SCR2RTF1: 45 pmole; SCR2RTR1: 45 pmole.

Figure 47 : Agarose gel electrophoresis of the RcSCR2 RT-PCR product after different amplification cycles. Total RNA of the endosperm of different days old seedlings was used as template to perform RT-PCR. The RcSCR2 was amplified and then checked in an agarose gel. A: 24 amplification cycles.

B: 28 amplification cycles. C: 32 amplification cycles. D: 36 amplification cycles. E: 40 amplification cycles. M: 100bp DNA marker; 1: amplification product of day 2; 2: amplification product of day 3; 3:

amplification product of day 4; 4: amplification product of day 5; 5: amplification product of day 6.

The first strand cDNA from reverse transcription was used as the template for the subsequent Real Time PCR. The PCR was performed as described in ‘Material and Methods’. It was performed in ABI-7000 system and the signal was collected.

Subsequently the data were analyzed and exported to Excel. The PCR product was also checked in an agarose gel. If non-specific amplification bands could be seen, the data could not be used. The experiment had then to be repeated from the reverse transcription step.

RcSCR2 transcripts were undetectable in the endosperm by RNA-blot hybridization, but low levels of RcSCR2 gene expression were detected after conventional RT-PCR and real time RT-PCR in the presence of RcSCR2-specific primers. Typical amplification plots for nsLTPc1 and RcSCR2 in the endosperm during real time RT-PCR analysis are shown in Figure 48 and Figure 49.

When the RcSCR2 transcript values in samples were normalized to the amount of the artificial control nsLTPc1 in samples, the lowest level of RcSCR2 mRNA expression was found in day 2 endosperm. Its value was set up as 1 and the RcSCR2 expression in other samples was calculated relative to the day 2 endosperm level (Figure 50). Expression of RcSCR2 for other days was also very low. The highest levels of RcSCR2 mRNA were observed in the day 3 endosperm sample, about 4-fold higher than in day 2 endosperm.

The results of Northern blot hybridization, RT-PCR and real time RT-PCR clearly show that the expression of RcSCR2 is very low.

Figure 48 : The real time RT-PCR fluorescence profile of the nsLTPc1.

Figure 49 : Real time quantitative RT-PCR analysis of RcSCR2 expression in endosperm. Quantitative RT-PCR was performed on total RNA isolated from different days old endosperm as described in Materials and Methods.

Figure 50 : Quantification of RcSCR2 expression in different days old endosperm. RcSCR2 expression values were normalized to the level of the amount of nsLTPc1 in different days samples.

RcSCR2 expression in day 2 endosperm was set up as 1.