In an attempt to obtain the most accurate measurement of Claire’s sheared, heat shock mantle DNA, I quantified the samples using a third method: fluorescence.
Samples were quantified using the Quant-It DNA BR Kit (Life Technologies/Invitrogen) according the manufacturer’s protocol. Standards were run in triplicate. Due to low sample volumes, only 1μL of each sample was used and was not replicated.
Plate was read on a Perkin Elmer plate reader using the Wallac software. The plate was measured three times, with each well measured for a one second duration on each read.
Results:
Spreadsheet: 20150303_gigasHSshearedDNApico
Comparison of NanoDrop1000, and fluorescence measurements:
| Sample | NanoDrop (ng/μL) | Bioanalyzer (ng/μL) | Fluorescence (ng/μL) |
| 2M sheared | 48.03 | 16.28 | 4.91 |
| 4M sheared | 190.96 | 58.52 | 48.10 |
| 6M sheared | 141.56 | 42.98 | 28.42 |
| 2MHS sheared | 221.93 | 32.45 | 13.48 |
| 4MHS sheared | 257.48 | 43.82 | 11.75 |
| 6MHS sheared | 202.02 | 51.12 | 8.97 |
Not entirely surprising, but the fluorescence method is clearly the most conservative measurement of the three methods. However, I do find the difference between the Bioanalyzer and fluorescence measurements very surprising. I suspected the Bioanalyzer would underestimate the concentrations because I actively selected which peak regions to measure, possibly leaving out some aspect of the sample.
Regardless, will use the most conservative measurements (fluorescence) for decision making.
With our yields, we have insufficient DNA to conduct MeDIP and then subsequent bisulfite conversion and library prep on our own. The recovery from the MeDIP will result in too little input DNA for bisulfite conversion and, in turn, library prep.
However, we do have sufficient quantities of starting DNA (>200ng) for Epigentek’s MeDIP Methyl-seq. I have contacted Epigentek to see if their procedure includes bisulfite conversion after MeDIP (which the website workflow suggests that it does not).