Frequently Asked Questions (FAQs)
Q: How can I submit my samples?
A: If possible, we suggest contacting us before experiment design and sample preparation. Once your samples are ready, please download and complete the Sample Submission Form. Then, email it to us and let us know the submission date. In this way, we can prepare for your project in advance. Please always download the form from our website to ensure you use the latest version.
Q: Do you have any suggestions for sample preparation?
A: Many RNA samples submitted to our core do not pass QC. Please contact us for suggestions specific to your project. We also provide an RNA extraction service.
Q: My total RNA samples are degraded and I do not have replacement samples. Can you still use them to perform RNA-seq and miRNA-seq?
A: Yes. With degraded RNA, we use alternative approaches for RNA-seq and miRNA-seq. However, more reads are required for analysis, and the cost could be higher.
Q: Which kit would you recommend for total RNA extraction?
A: Basically, all major brand kits have good performance for total RNA extraction. However, taken the yield, quality and further application into consideration, we recommend using mirVana miRNA isolation kit (Lifetech), and suggest following the manufacturer’s total RNA extraction protocol to extract RNA from culture cells and most types of tissues. In this way, the extracted total RNA can be used for both RNA and small RNA study. Please be reminded that for some special samples such as blood and FFPE samples, special kits may be required.
Q: Why are my RNA samples extracted from blood and other tissues degraded even when I flash-frozen them in liquid nitrogen and stored them in -80C freezer?
A: It is very important to treat blood samples according to established protocols before storing them for future RNA extraction. GESC suggests our customers to strictly follow the Invitrogen’s protocol by mixing the anticoagulated whole blood with RNAlater, or using other commercially available kits specifically for this purpose. For most types of fresh tissues, it is suggested to treat them with RNAlater rather than liquid nitrogen.
Q: Will genomic DNA contamination affect my RNA-seq data quality?
A: If your RNA extraction protocol has a phase separation step, please make sure that the interphase which contains gDNA is not disturbed when transferring the aqueous phase. If you have enough RNA, it is always good to leave some aqueous phase. Specifically for Ribo-Zero RNA-seq, please treat the RNA with RNase-free DNase I; for polyA RNA-seq, there is less concern about gDNA contamination, as the protocol only purifies polyA RNA for sequencing.
Q: In regard to your routine polyA RNA-seq, how much total RNA should I submit, what should the concentration be, and what should the quality be?
A: We run our routine polyA RNA-seq with the total RNA input between 50 ng – 1 ug in 50 ul. In general, the RNA quality determined by Bioanalyzer RIN (RNA Integrity Number) score should be >7. Please adjust all the RNA samples to the same 100 ng/ul (preferred concentration) and submit 1.5- 2 ug RNA, if possible. Please do not adjust the RNA concentration if it is < 20 ng/ul.
Q: Can you give me an exact sequencing turnaround time?
A: While our goal is to provide short turnaround time, many factors affect it. One consideration is that we have to fill an entire flow cell with the expected number of samples before the final sequencing. As our service began to expand since 2012, there has been a reduction in turnaround time.
For single read sequencing (SR 1x50 bp), our goal is to keep the turnaround time within one month. The pair-end sequencing takes longer time and is more difficult to predict the turnaround time. Please contact us for current turnaround time. Please note bioinformatics analysis takes extra time.
Q: I want to compare my two groups. How should I specify this?
A: If you want to compare two groups (group A and group B), and you want to set group A (such as a Control group) as a reference to see the change in group B (such as a Treatment group), please tell our bioinformatics team to "compare group B with group A", not "compare group A with group B". The calculation is always the first mentioned group over the 2nd mentioned group.
Q: What can I expect from the bioinformatics analysis?
A: After the completion of the sequencing, and if the data pass preliminary QC analysis, we will contact you for sample information. This includes 1) how many groups do you have? 2) the sample IDs in each group; 3) are they paired samples or not? 4) how do you want us to compare different groups? Once we have the information, our bioinformatics team will perform standard analysis, and send you a link to the result.
Q: What are your preferred payment methods?
A: For payment from UC, please provide the PI’s full grant account string (e.g. G100121-62612XXXXX-1-100XXXX); for payment from VA, only credit card payment is accepted. We will contact you for credit card information. For payment from institutions other than UC and VA, please mark your check with GESC order/invoice #, and mail it to Ben according to the instruction provided in the invoice.
Q: My grant account will be expired soon. Can you help me to process the payment now?
A: Yes. Please contact us as soon as possible.
Q: How will you adjust your service rate?
A: Our service rate is reviewed and approved by the Recharge Council. The rate heavily relies on the cost of sequencing consumables and other reagents. While it is our goal to maintain our services at low stable rate, occasionally we may adjust our rate without prior notice based on price changes in the market.
Supporting letter, publication, collaboration and method development
Q: Do you provide support for grant and manuscript writing?
A: Yes. We provide free supporting letter, service quotes, and our standard Materials & Methods for grant preparation/manuscript writing. We also provide consultation on experiment design and suggestions for sample preparation.
Q: How about collaboration?
A: We are very interested in collaboration. With our expertise in the field, we are looking for collaboration opportunities in grant application and paper submission. Specifically, if the study needs to establish a novel approach, needs to use a complicated, customized method or extensively requires our expertise, collaboration is required.
Q: Are you interested in method development?
A: Yes we are very interested in developing or introducing new methods to our core that can benefit both the research community and us. Please contact us if you think we should provide a service that is not on our list.
Q: I noticed you changed your address?
A: The Postal Service assigned our building a new address (160 Panzeca Way, Kettering Building Room 336, Cincinnati, OH 45267). However, our physical location remains the same.
Department of Environmental & Public Health Sciences
Kettering Lab Building
160 Panzeca Way
Cincinnati, OH 45267-0056
Mail Location: 0056