Preparing Samples for RNA Sequencing
Cofactor’s standard pipeline accepts total RNA extracted from your organism of interest. If your project does not currently include RNA extraction, and you would like us to start from tissue, cell pellets, FFPE, or any other biological material, please contact [email protected] to ensure this is added to your project quote.
The key to a successful RNA-seq experiment is starting with a sufficient quantity of high quality material. For most RNA extractions, we recommend the mirVana extraction kit from Ambion or the RNeasy kit from Qiagen, however, you may use your own lab protocols or equivalent kits if they have yielded high quality RNA in the past. Small RNA sequencing also requires total RNA as starting material, however, the extractions must be modified to allow retention of small RNAs.
All RNA extractions must also be accompanied by a DNase treatment. Qiagen protocols allow this step to be performed on the column. For guidance with DNase treatment using other kits and protocols please contact us at [email protected]. Finally, all RNA samples must be re-suspended in nuclease-free water and shipped frozen on dry ice.
Assessing Sample Quantity and Quality
Please measure RNA concentration with a fluorescence-based measurement such as a Qubit or Picogreen assay. These measurements tend to be specific for RNA and are preferable to NanoDrop or other UV-based measurements which also measure contaminating genomic DNA. If your lab does not have a way of making a fluorescence-based measurement, please use whatever method is available and indicate this information when submitting your sample. For NanoDrop measurements, samples must fulfill the requirements below:
UV absorption: 260/280 > 1.9 and 260/230 > 2.0
RNA sample quality is best assessed on an Agilent Bioanalyzer or equivalent instrument (see example traces on next page). Alternatively, if a Bioanalyzer is not available, please run the RNA samples on a denaturing agarose gel to confirm the integrity of rRNA peaks.
The Bioanalyzer will produce an RNA Integrity Number (RIN) which is an objective measure of RNA quality. RIN scores vary from 1-10 with 10 being the highest quality samples showing the least degradation. RINs for all samples (except from FFPE, as described below) should ideally be >7. For some sample types such as RNA from insects, the Bioanalyzer RIN may not be a reliable measure of RNA quality if the organism does not show conventional eukaryotic or prokaryotic ribosomal RNA peaks. Please contact us at [email protected] for assistance in assessing the quality of these samples.
RNA analysis results obtained with the Agilent 2100 Bioanalyzer and the Agilent 2200 TapeStation are displayed in form of a gel image, electropherograms and in a tabular format. RNA quality assessment is based on the RNA Integrity Number (RIN).
Image source: Agilent Technologies.
FFPE samples are another exception to the sample quality requirements since the RNA from these is heavily fragmented. Often RIN scores for fragmented RNA are misleading. Do not be concerned if the RIN scores for these samples are significantly below the threshold. We will assess quality upon arrival at Cofactor.
Samples for Total RNAble, RNAccess and RNAmplify may also be lower RIN values. Ideally, you have measured this quality information prior to deciding upon a final experimental design with your Project Scientist.
How many cells or how much tissue do we need for sequencing?
A figure summarizing the minimum RNA quantity and quality may be found on our Discovery Services page.
The following are minimum starting tissue requirements for RNA isolation, library construction, and QC. If you don’t see your project listed below, please contact [email protected]. If your samples do not meet the minimum requirements described below, please contact [email protected] ahead of sample submission to confirm samples can be accommodated.
|Product Name||Catalog Numbers||Library Type||Minimum Amount of Tissue|
|mRNAble||CFG001-CUSTOM||Standard mRNA Sequencing (poly-A enrichment)||10-30 mg of tissue, 5 mL bacterial culture pellet, 106 mammalian cell pellet|
|Total RNAble*||CFG002-CUSTOM||Standard mRNA Sequencing (poly-A enrichment)||10-30 mg of tissue, 5 mL bacterial culture pellet, 106 mammalian cell pellet|
|FFPExact*||CFG004-CUSTOM||Fragmented RNA Sequencing (whole transcriptome/rRNA depleted)||4 each 5-10 µm FFPE sections or 5-10 mg tissue‡|
|miRNAble||CFG002-CUSTOM||miRNA enrichment||10-30 mg of tissue, 5 mL bacterial culture pellet, 106 mammalian cell pellet|
|RNAccess¥||CFG006-CUSTOM||Low-input/low-quality (targeted) RNA Sequencing||2 each 5-10 µm FFPE sections or 5-10 mg tissue‡|
|RNAdvantage¥||CFG008-CUSTOM||Comparative Expression + Fusion Detection||2 each 5-10 µm FFPE sections or 5-10 mg tissue‡|
|RNAssemble||CFG010-CUSTOM||Normalized mRNA Sequencing (poly-A + DSN)||10-30 mg of tissue, 5 mL bacterial culture pellet, 106 mammalian cell pellet|
|Custom Protocol||CFG000-CUSTOM||Custom Protocol||Contact Us|
|R&D Protocol||CFG000-R&D||R&D Protocol||Contact Us|
*Total RNAble and FFPExact are specifically for RNA derived from human, mouse, and rat samples.¥RNAccess and RNAdvantage are only for RNA derived from human samples. All other products/catalog numbers are amenable to any organism. If a description for your sample type is not listed, please contact [email protected] for guidance on the amount of material to ship.
‡Some tissues such as epidermal tissue may require larger amounts. Contact your Project Scientist for guidance.
How much RNA do we need for sequencing?
The table below contains minimum starting requirements for RNA isolation, library construction, and QC. If you don’t see your project listed below, please contact [email protected]. If your samples do not meet the minimum requirements described below, please contact [email protected] ahead of sample submission to confirm samples can be accommodated.
|Product Name||Catalog Numbers||Library Type||Recommended RNA Quantity‡||Minimum RNA Concentration‡|
|mRNAble||CFG001-CUSTOM||Standard mRNA Sequencing (poly-A enrichment)||≥500 ng||≥5 ng/µl|
|Total RNAble*||CFG002-CUSTOM||Standard mRNA Sequencing (poly-A enrichment)||≥500 ng||≥25 ng/µl|
|picoRNA||CFG003-CUSTOM||Low-input/high-quality mRNA Sequencing (poly-A enrichment)||≥100 pg or equivalent cell number**||≥100 pg/µl|
|FFPExact*||CFG004-CUSTOM||Fragmented RNA Sequencing (whole transcriptome/rRNA depleted)||≥500 ng or equivalent FFPE sections†||≥25 ng/µl|
|miRNAble||CFG002-CUSTOM||miRNA enrichment||≥1 µg||≥100 ng/µl|
|RNAccess¥||CFG006-CUSTOM||Low-input/low-quality (targeted) RNA Sequencing||≥100 ng||≥20 ng/µl|
|RNAmplify||CFG007-40, CFG007-70, or CFG007-CUSTOM||Low-input/low-quality (targeted) RNA Sequencing||Contact Us||Contact Us|
|RNAdvantage¥||CFG008-80 or CFG008-CUSTOM||Comparative Expression + Fusion Detection||≥100 ng||≥20 ng/µl|
|RNAssemble||CFG010-CUSTOM||Normalized mRNA Sequencing (poly-A + DSN)||≥1 µg||≥5 ng/µl|
|RiboPRO||CFG011-CUSTOM||Ribosomal Profiling||Contact Us||Contact Us|
|All_Splice||CFG012-CUSTOM||PacBio Long-Read RNA Sequencing||Contact Us||Contact Us|
|Custom Protocol||CFG000-CUSTOM||Custom Protocol||Contact Us||Contact Us|
|R&D Protcol||CFG000-R&D||R&D Protcol||Contact Us||Contact Us|
‡Quantities listed above represent Qubit quantified material; RNA quantified using NanoDrop or other absorbance methods will require additional material.
*Total RNAble and FFPExact are specifically for RNA derived from human, mouse, and rat samples.¥RNAccess and RNAdvantage are only for RNA derived from human samples. All other products/catalog numbers are amenable to any organism.
**Equivalent cell number is required for sorted cells shipped to Cofactor in extraction buffer. Contact [email protected] for more information on this method.
†Equivalent FFPE sections based on average RNA extraction yield.
Sample Submission Guidelines Checklist
At the time of sample submission, please provide the genomic or transcriptomic reference sequence to which your sequence data will be aligned. Reference sequences can be submitted by email to [email protected] as a FASTA file or a URL where the FASTA can be downloaded.
If your experimental design allows, we recommend shipping additional samples as “backup” in case of sample QC failure. Please note which samples are considered “backup” on the Sample Submission Form.
Pictures and/or Bioanalyzer traces can also be emailed ahead to avoid delays due to poor-quality samples. Send to [email protected].
Please use the following checklist for sample submission:
- RNA is DNase treated and is re-suspended in nuclease-free water.
- Sample quality, quantity, and concentration pass above thresholds.
- All samples are submitted in 1.5 ml micro-centrifuge tubes clearly labeled with sample names matching the Sample Submission Form, preferably tubes are sealed with parafilm.
- Fresh tissue and RNA is frozen and will be shipped on plenty of dry ice. Avoid shipping samples on Fridays to keep samples from prolonged exposure to ambient temperature.
- Sample submission form completed online at https://cofactorgenomics.com/sample-submission. Please attach the sample list spreadsheet to the online submission form electronically.
- Printed copy of the sample list spreadsheet is placed in the package.
- Email a pdf copy of your signed project quote to [email protected].
- Genomic or transcriptomic reference sequence is sent to [email protected].
Don’t forget to go back to the Sample Submission Page to submit your Excel Spreadsheet and other information.
International customers, please refer to the “Guidelines for International Shipments” document for additional shipping instructions.
Still have questions? Send us an email at [email protected] or give us a call at 314-531-4647 and we’ll be happy to help you!