| Sample preparation options | RNA Amount | Concentration | Minimal volume |
|---|---|---|---|
| INCPM mRNASeq | 500 ng | 20 ng/μl and above | 5 μl *** |
| INCPM mRNASeq w/o polyA selection* | 80 ng | 13 ng/μl and above | 6 μl |
| Bulk MARS-seq | 1-60 ng | 10 ng/μl ** | 6.1 μl |
| Illumina TruSeq Stranded mRNA | 0.1 - 4 μg | 5-50 μl | |
| Bulk SMART-seq 2 | 0.1-50 ng or 100 cells/nuclei | Max volume 4.6 μl |
- If more than 16 samples are submitted, we recommend to bring all samples to the same concentration in a v-shaped 96-well plate (PCR plate, column wise). If submitting more than 24 samples,submission in a plate is required.
* For RNA samples that do not contain polyA, please perform ribosomal depletion or contact us.
** For submitting RNA for bulkMARS-seq see guidlines here.
*** Note that final amount needs to be above 500 ng. If submitting samples in a plate, submit the same concentration for all samples.
NanoString
NanoString is a probe-based technology used to measure gene expression of a few hundred genes per sample. It may fit samples that do not fit RNA-seq (for example, FFPE samples). There are panels for gene expression and panels for miRNA expression.
The requirements for sample submission are:
- Each NanoString kit is suitable for 12 samples. Once we thaw the kit we must use it, and cannot use leftovers at another time. Therefore it is best to plan experiments of 12 samples (or multiplication of 12 samples).
- The researcher may use any RNA extraction kit that works well for their type of samples. When submitting samples for miRNA analysis, the RNA extraction kit should extract also miRNA.
- There are specific instructions for collecting RNA from plasma – please contact us.
- The RNA should have OD260/280>1.9 and 260/230>1.8
- We highly recommend running a TapeStation to measure the DV200 (the percentage of RNA fragments at a length of 200 or more), and the value should be 50% and up. If the values are lower the probes might not bind efficiently.
- For gene expression panels – bring 10ul from each sample at a concentration of 30ng/ul
- For miRNA panels – bring 6ul from each sample at a concentration of 33ng/ul.
- Please submit twice the amount from that noted in the table
- Please add extra volume to enable accurate pippettation
- Measure RNA concentration by nanodrop or Qubit (do not rely on Bioanalyser or Tapestation concentration).
| Quality parameters | RNA |
|---|---|
| OD260/280 | ≥ 1.8 |
| OD260/230 | ≥ 2 |
| RIN number | ≥ 8 |
- Measure your RNA concentration by nanodrop or Qubit. If RNA concentration is below ~40ng/ul, we recommend to measure it by Qubit which is more accurate.
- Run your RNA on a TapeStation, Bioanalyzer or gel to ensure high integrity. If you need a service for TapeStation analysis for the RNA, please contact Muriel Chemla, 089344435, muriel.chemla@weizmann.ac.il.
- Please note that Agilent RIN numbers are based on human, mouse, and rat RNA profiles. Other organisms’ profiles may not generate high RIN numbers, but may still be high quality. You should find the expected profile for your species and assess the quality.
- RNA samples that underwent polyA selection or ribosomal depletion cannot be evaluated for RIN score (as it is based on ribosomal RNA)
- Note that the user is responsible for the results in case the sample does not match the specifications above, or do not meet the criteria of the procedure requested.