Table I
Primers used in the RT-qPCR.
| Target | Forward Primer | Reverse Primer |
|---|---|---|
| 1000-A | 5’-CAACCGGTGTGATCAGGACA-3’ | 5’-AGGACAGTCCGCATAAGCAC-3’ |
| 1000-B | 5’-TACCAGCGCTTCTGTACGAC-3’ | 5’-GAGCTGTATCCGTGCCGTAA-3’ |
| 500-A | 5’-TCGCAGGCCTAATACGTGTC-3’ | 5’-CGTGAATCTCGGAGCGGTAA-3’ |
| 500-B 3’end | 5’-GGGTAGCGATTTAACGACTCG-3’ | 5’-CAGAGCCTGCCTTATCGTGA-3’ |
| 500-B middle | 5’-CCGAACGCTACGTGACGATA-3’ | 5’-ATCTACATGTTCCGTGCGCA-3’ |
| 500-B 5’end | 5’-AGACTAAATCTCGGCGTCGG-3’ | 5’-TAGATAGGGTCCGCATGACG-3’ |
| 500-C 3’end | 5’-GCACGACCGAATTATGCACC-3’ | 5’-AACCACTGACGTGAGCGATT-3’ |
| 500-C middle | 5’-TAGACGCGCCTTACTCCTCT-3’ | 5’-TAGTGGAGCTCGCGGATTTG-3’ |
| 500-C 5’end | 5’-GGACTAAACGCACTGAATACCG-3’ | 5’-ATCGCCCGTACTATCCGGTA-3’ |
Fig. 1.
RNA yields from S. cerevisiae and E. coli with hot phenol RNA extraction method using standard RNAs. Standard RNA yields were calculated based on the RT-qPCR result. Cell disruption (using phenol and SDS) was evaluated using standard RNA 1000-A. Purification (using chloroform) was evaluated using standard RNA 1000-B. Precipitation (using ethanol) was evaluated using standard RNA 500-A.
Table II
Inhibition of RT-qPCR by RNA extract solutions from S. cerevisiae and E. coli were evaluated using standard RNAs (%).
| Species | 1000-A | 1000-B | 500-A |
|---|---|---|---|
| S. cerevisiae | –1.1 ± 4.8 | 50.4 ± 4.0 | 0.3 ± 3.1 |
| E. coli | 7.0 ± 3.2 | 44.6 ± 3.0 | 27.7 ± 2.8 |
Fig. 2.
Degradation of standard RNAs 500-B or 500-C by S. cerevisiae or E. coli crude extract.
Fig. 3.
The different degree of RNA degradations depends on the RNA regions in S. cerevisiae crude extract. Standard RNAs were degraded by E. coli crude extract at 30 min and by S. cerevisiae crude extract at 20 min. *, p < 0.001, n = 3, t-test.
Table III
Survival of 3’ end and 5’ end and the ratio of 3’ end to 5’ end regions of standard RNAs after degradation with S. cerevisiae RNA crude extract.
| Standard RNA | 500-B | 500-C | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Degradation time (min) | 0 | 10 | 20 | 60 | 180 | 0 | 10 | 20 | 60 | 180 |
| 3’ end (survival %) | 100 | 73.5 | 45.2 | 32.1 | 6.3 | 100 | 76 | 59.2 | 26.7 | 3.2 |
| 5’ end (survival %) | 100 | 85.9 | 71.2 | 67.9 | 6.4 | 100 | 88.9 | 92.9 | 67.2 | 8.6 |
| 3’ end /5’ end (ratio) | 1 | 0.86 | 0.63 | 0.47 | 0.99 | 1 | 0.86 | 0.64 | 0.4 | 0.37 |