The ability to measure nucleic acid concentration, purity and integrity is critical, as it is the starting point of every assay that we perform. The Ramaciotti Centre employs the following methods:
1. Quantification and Purity Assessment
Samples are read on a spectrophotomer to assess concentration and purity. Samples can fail this QC step for several reasons:
- sample too dilute, concentration lower than requested
- sample far too concentrated
- sample contaminated with protein, 260:280 ratio less than 1.8
- samples contaminated with reagents used during the extraction protocol, 260:230 ratio less than 1.8
Pure RNA should have a A260:A280 ratio between 1.8–2.1 and a A260:A230 ratio of 1.8–2.3. Ratios of less than 1.8 indicate contamination with proteins or chemicals used in the extraction procedure, or dilution of your samples in full strength TE. In most cases samples with a 260:230 ratio of less than 1.0 are OK. However as we cannot determine the nature of the contaminant we cannot guarantee it will not interfere with the labeling or library preparation procedure.
2. Additional Quantification Assessment by Fluorescent Assay
In addition to being read on a spectrophotometer, all samples that are submitted for next-generation sequencing are routinely quantified using a fluorescence-based assay such as RiboGreen, PicoGreen or Qubit. Samples for other services that are too low in concentration to be reliably quantified using a spectrophotomer, are also quantified using a fluorescence based assay.
3. Integrity Check
Total RNA – Agilent Bioanalyzer & TapeStation
Total RNA samples are run on either the Agilent Bioanalyzer or TapeStation to assess the integrity of the total RNA. An RNA Integrity Number is produced for each sample. RNA extracted from poor quality tissue samples or cells that has degraded during extraction is a poor candidate for microarray or next-generation analysis and should not be used. The final decision on whether to process a sample rests with the customer. Customers can assess whether their RNA is of good quality by searching the RIN database (RINdb) to view a “normal” RNA profile for their tissue.
Genomic DNA – Agilent TapeStation & Perkin Elmer LabChip GX
The integrity of genomic DNA is assessed using the Agilent TapeStation or Perkin Elmer LabChip GX. This provides a DNA Intergrity Number (DIN) or Genomic DNA Quality Score (GQS), respectively. This is calculated from the size distribution of the sample.
Amplicons – Agilent TapeStation & Bioanalyzer
Amplicons submitted for sequencing are run on the TapeStation or Bioanalyzer to accurately the check size.
For further information on interpreting QC data please download our RAMAC_QC_Datasheet.
Important Note on Quality Control
If a sample fails initial quality control the customer can resubmit replacement samples or withdraw their submission. If the customer chooses to resubmit their samples they must do so within a 6 week period. Sample submission after the 6 week period has elapsed will be considered as a new submission. If the customer withdraws their samples they must pay for any consumables ordered on their behalf. The Centre can only guarantee high quality data if procedures are carried out with good quality nucleic acid. Poor quality nucleic acid will produce sub-optimal results. The decision on whether to process a sample remains with the customer. In the case where a poor quality sample is processed, the facility cannot guarantee the outcome and the customer must accept responsibility.