- What is the significance of the 260/230 ratio in my SAQC request?
- What is the best protocol to extract RNA from tissues?
- Should I use total RNA or polyA RNA for my reactions?
- What is the minimum requirement for RNA quantity?
- Do I need to bring the spectrophotometer or gel information for my RNA samples?
- Can I use amplified RNA (aRNA) for my experiments?
- What do I need to do when I am dropping off samples for full service and/or quality assurance?
- What do I need to bring for the Affymetrix basic service?
- How many replicate arrays do I need to do per sample?
Q: What is the significance of the 260/230 ratio in SAQC?
A: A low 260/230 ratio may indicate contamination of organic or carbohydrate origin. More information can be found on the .pdf file Significance of the 260/230 Ratio.
Q: What is the best protocol to extract RNA from tissues?
A: There is not a 'best' protocol to isolate and extract RNA from tissues, but we have found that the RNeasy Kits from Qiagen produce very good results. Although it is not necessary, we also strongly suggest using the optional On-Column DNase Digestion found in Appendix D of the complete protocol. Genomic DNA contamination of RNA samples can cause an increase in false positives. The protocol is also available at the Qiagen web site.
Note: Users who are using very small amounts of tissue and/or cells can use the RNeasy Micro Kit from Qiagen.
Q: Should I use total RNA or polyA RNA for my reactions?
A: Either one is okay, but we suggest using total RNA for Affymetrix, Agilent, and NimbleGen protocols.
Q: What is the minimum requirement for RNA quantity?
A: Affymetrix GeneChips® 3'IVT protocol: 100 ng of total RNA in a volume of 3.0 µL for Affymetrix experiments. Affymetrix GeneChips® Exon and Affymetrix GeneChips® Gene ST 1.0 protocol: 1ug of total RNA in 3.2uL (minimum concentration 313ng/uL). Affymetrix GeneChips® microRNA protocol: 1 ug of total RNA in 8uL (minimum concentration125ng/uL)
Agilent Expression Arrays: 1ug of total RNA in 8.3uL (minimum concentration 120ng/uL). Agilent microRNA Arrays: 100ng of total RNA in 4uL (minimum concentration 25ng/uL).
NimbleGen Eukaryotic protocol: 10ug of total RNA in 10uL (minimum concentration 1000ng/uL). NimbleGen Prokaryotic protocol: 1ug of total RNA in 10uL (minimum concentration 100ng/uL).
Note: Users wishing to use aRNA (amplified RNA), should READ and UNDERSTAND the protocol that they plan to use and that they have VERY GOOD experimental design in order to end up with usable material for submission (i.e. labeled appropriately). Users who wish to do amplification of small quantities of RNA should meet with MCF before starting their experiment to make sure their experimental design is appropriate.
Q: Do I need to bring the spectrophotometer info or gel image for my RNA samples?
A: No, GARP does not need this information. Users can use this information to help decide what dilution factor to provide us with for the initial Agilent submission, but we do not use the gel/spec info to decide quality of RNA for microarray experiments. We use the Agilent 2100 Bioanalyzer and the NanoDrop ND-1000 Spectrophotometer as part of our SAQC service.
Q: Can I use amplified RNA (aRNA) for my experiments?
A: Yes, you can. We have small sample protocols for cDNA experiments and Affymetrix experiments where aRNA can be used when your RNA quantity available is too low. However, we also have alternative protocols for total RNA when there is too little starting material for the standard protocol. (Please see alternative protocol section).
Note: Please contact us prior to starting your experiment if you plan to use aRNA for any single-stranded probe hybridization (i.e. Affymetrix GeneChips®, oligonucleotide arrays, etc.)
Q: What do I need to do when I am dropping off samples for full service and/or quality assurance?
A: When dropping off any samples to GARP, an SAQC request should already have been submitted to MAID and the user should receive a confirmatory email instructing them to bring the samples for that request to the laboratory. Each user must bring 5.0-6.0 µL of the diluted RNA samples they wish to run at a concentration range of 25-500 ng/µL, or 200-5000 pg/µL for small samples. The bioanalyzer's range of detection for RNA is 25-500 ng for the Nano LabChip or 200-5000 pg for the Pico LabChip, and we need 2.0 µL to run the assay. We also run 1.5µL your sample on the NanoDrop® ND-1000 Spectrophotometer to get an accurate concentration of your sample. We suggest you bring at least 5.0-6.0 µL per sample for error and in case multiple runs are needed.
Genomic DNA samples for Array-CGH or the Affymetrix SNP Mapping GeneChips should be submitted as an SAQC first. These samples will be tested using the NanoDrop ND-1000 Spectrophotometer and run on a 2% agarose gel to test for integrity.
Please bring all samples in 1.5 ml tubes and put sample ID numbers on the top of the corresponding tubes. (We will not accept 0.6 ml or 0.2 ml tubes.)
Note: After GARP has judged the quality of the material, results of the completed assay will be accessible to the user via MAID. The information should be used to prepare the RNA or DNA sample for Affymetrix or cDNA/Spotted Array submission. In order to open a result file emailed to you by the GARP (also accessible via MAID), you will need to download and install the Agilent Expert Review Software for Molecular Assays (DNA/RNA), available for free from the Agilent web site.
Q: What do I need to bring for the Affymetrix hybridization service?
A: Some users request only hybridization, staining/washing, or scanning services with GARP. The samples to be hybridized should first be submitted as an SAQC on MAID with information in the notes section to alert the technicians that this sample will by labeled by the user and only hybridized, stained, and scanned in GARP. Each sample submitted for Affymetrix basic service should be biotinylated cRNA. GARP will quantify the sample for concentration and fragment the cRNA. We will then prepare the sample(s) for hybridization, staining, washing, and scan the GeneChip®. If the user intends to provide their own GeneChips® they need to bring them to the MCF as well.
Q: How many replicate arrays do I need to do per sample?
A: It is always up to each individual researcher how many replicate arrays they do. Users can use the same sample on different arrays, or they can use the same treatment on different arrays, even if the samples are not from the same RNA extraction. GARP does require at least 2 replicates per sample for all Full Service Experiments, and recommends 3 replicates per sample.
Please note: The same sample on different arrays is considered a technical replicate, but the same treatment on different arrays is a biological replicate. Remember, the more replicates you have, the more accurate, reliable, and believable your data will be.