RNA quality check after PCR inhibitors removal - Hawai TPC
RNA quality check before and after removal of potential PCR inhibitors - Pocillopora acuta, Montipora capitata, Porites compressa, Hawaii TPC project
Protocols used
Here I’m checking the quality of 6 RNA samples (n=2 per species, Pocillopora acuta, Montipora capitata, Porites compressa) by TapeStation, Nanodrop and QBIT before and after using the Zymo OneStep PCR Inhibitor Removal Kit. Library prep hasn’t been very successful, see summary, so we want to test for the presence of potential PCR inhibitors. I’m using the same input for all samples, 759 ng in 55 ul (the kit wants a minimum of 50 ul).
Qubit
| sample_id | Species | Temp | RNA_AVG (ng/ul) before | RNA_AVG (ng/ul) after | ||||
| Pcom-G10 | Porites compressa | 26.8 | 51.8 | 13.0 | ||||
| Pcom-F8 | Porites compressa | 25 | 17.9 | 17.8 | ||||
| Pacu-H1 | Pocillopora acuta | 18 | 20.5 | 11.5 | ||||
| Pacu-D8 | Pocillopora acuta | 25 | 19.3 | 13.3 | ||||
| Mcap-G1 | Montipora capitata | 18 | 53.9 | 12.7 | ||||
| Mcap-F10 | Montipora capitata | 26.8 | 13.8 | 12.5 |
before PCR inhibitors removal - after PCR inhibitors removal
Nanodrop
- Expected 260/230 values are commonly in the range of 2.0-2.2. If the ratios are lower or abnormally higher it indicates the contamination of the samples with certain protein or phenolic compounds
- A 260/280 ratio of around 2.0 is generally considered pure for RNA. Abnormal 260/280 ratios usually indicate that a sample is contaminated by residual phenol, guanidine, or other reagent used in the extraction protocol
| sample_id | Species | Temp | RNA_AVG (ng/ul) before | RNA_AVG (ng/ul) after | 260/230 before | 260/230 after | 260/280 before | 260/280 after | ||||||||
| Pcom-G10 | Porites compressa | 26.8 | 24.35 | 19.2 | 1.08 | 0.88 | 3.03 | 2.24 | ||||||||
| Pcom-F8 | Porites compressa | 25 | 26.1 | 25.25 | 3.88 | 1.03 | 2.99 | 2.33 | ||||||||
| Pacu-H1 | Pocillopora acuta | 18 | 20.1 | 12.95 | 0.76 | 0.59 | 3.30 | 2.72 | ||||||||
| Pacu-D8 | Pocillopora acuta | 25 | 12.1 | 31.3 | 0.99 | 0.68 | 3.96 | 2.70 | ||||||||
| Mcap-G1 | Montipora capitata | 18 | 28.96 | 15.45 | 3.65 | 0.95 | 3.11 | 2.58 | ||||||||
| Mcap-F10 | Montipora capitata | 26.8 | 12.6 | 20.65 | 1.67 | 0.73 | 5.45 | 2.89 |
Nanodrop results before the cleanup and previous library prep attempt
I’m putting Nanodrop results right next to my previous library prep attempts to look for potential patterns in the 260 ratios and library prep concentration.
| sample_id | Species | Temp | RNA_AVG (ng/ul) before | 260/230 before | 260/280 before | library QBIT (ng/ul) | ||||||
| Pcom-G10 | Porites compressa | 26.8 | 24.35 | 1.08 | 3.03 | 1.21 | ||||||
| Pcom-F8 | Porites compressa | 25 | 26.1 | 3.88 | 2.99 | 3.15 | ||||||
| Pacu-H1 | Pocillopora acuta | 18 | 20.1 | 0.76 | 3.30 | 0.43 | ||||||
| Pacu-D8 | Pocillopora acuta | 25 | 12.1 | 0.99 | 3.96 | not prepped yet | ||||||
| Mcap-G1 | Montipora capitata | 18 | 28.96 | 3.65 | 3.11 | 0.7 | ||||||
| Mcap-F10 | Montipora capitata | 26.8 | 12.6 | 1.67 | 5.45 | 1.1 |
TapeStation
| sample_id | Species | Temp | RNA_conc (ng/ul) before | RNA_conc (ng/ul) after | RIN before | RIN after | ||||||
| Pcom-G10 | Porites compressa | 26.8 | 28.5 | 13.0 | 9 | 8.4 | ||||||
| Pcom-F8 | Porites compressa | 25 | 21.5 | 20.8 | 8.4 | 8.7 | ||||||
| Pacu-H1 | Pocillopora acuta | 18 | 23.2 | 11.3 | 8.8 | 8.1 | ||||||
| Pacu-D8 | Pocillopora acuta | 25 | 28.0 | 14.6 | 9 | 8.4 | ||||||
| Mcap-G1 | Montipora capitata | 18 | 40.4 | 13.0 | 9.5 | 8.5 | ||||||
| Mcap-F10 | Montipora capitata | 26.8 | 16.8 | 13.0 | 8.1 | 7.9 |
before PCR inhibitors removal (first tape run per sample in the image below) - after PCR inhibitors removal (second tape run per sample in the image below)
