Genome Sequencing & Analysis Core Resource

Frequently Asked Questions

Shipping samples

Question:

" How do I ship the RNA to you?"

Response:

Ship the RNA on dry ice in a 1.5 ml Eppendorf tube, labeled with a 3- or 4- digit sample name.  Parafilm the tube cap so it does not pop open during shipment, and pad the tube so the plastic does not break at low temperature.  If you put tape on your tube, the adhesive will fail at dry ice temperature, and the tape will come off.  Inside your shipment, provide documentation so that we can tell when we open the cooler who the sample is from, your order #, which sequencing platform is needed, what are the sample names, and if you are sending total RNA, gDNA, your own library, etc. 

Question:

" What is your shipping address?"

Response:

Duke IGSP Genome Sequencing & Analysis Core Resource

ATTN: 454 Sequencing Service

130 Science Drive,

BSB Room 119 Durham,

NC 27708 USA

Question:

" I am shipping samples from abroad and would prefer not to send liquid samples, what do you recommend?"

Response:

We have heard about dry nucleic acid storage from Biomatrica, but have not validated it in our lab. We are willing to help you try it out by testing the integrity of your RNA when it arrives.  Send us reconstitution instructions.

Roche 454 questions: RNA samples

Question:

"What extraction methods do you recommend for RNA?"

Response:

We recommend you use liquid nitrogen and a tissuelyzer or genogrinder on tissue, followed by a Qiagen RNeasy kit or MoBio.

Question:

"How much total RNA do I need for 454 sequencing?"

Response:

30-75 micrograms total RNA in 50 microliters nuclease-free water.

Question:

"Why do I need so much RNA?"

Response:

454 library preparation does not involve amplification of the final library, therefore, more RNA is needed for 454 sequencing than for short-read sequencing. Additionally, the oligodT reaction we perform on the RNA has a very low yield, 0.5-1%, and 200 ng mRNA is required for the rest of the protocol.

Question:

" I think I can get higher yield out of an rRNA depletion reaction.  Can I send you 200 ng mRNA?"

Response:

We are concerned about the stability of mRNA, and never store mRNA after our oligodT reaction. mRNA goes straight into fragmentation and transcription in our lab. You are welcome to send your mRNA, but there is definitevely a risk that the library prep fails.