Sample and Documentation Requirements for the submission of EACH RNA sample

Use any conventional method or commercially available RNA isolation kits to extract RNA. Separate isolation of mRNA or microRNA from Total RNA is optional; Total RNA will be accepted. If submitting Total RNA for microRNA library preparation, use a method/kit that is designed for preserving the small RNA population in the Total RNA such as Ambion's mirVana miRNA isolation kit(cat#AM1560) or Qiagen's miRNeasy MiniKit (cat#217004). Kits will vary depending on the nature of starting material-cell, frozen tissue, FFPE tissue, etc. Please check with the manufacturer prior to use to ensure that it is compatible with your sample type. If less RNA is available than the quantity specified below or if the quality is poorer than what is specified, please contact us to discuss alternatives. All RNA samples should be resuspended in Nuclease-free, DEPC Treated water. Be sure to include Completed RNA Sample Submission Form with each sample submission.

Total RNA Sample
Sample Quality Best Option: RNA Integrity Number (RIN) ≥ 8, determined using an Agilent 2100 Bioanalyzer (28S peak>18S peak and 28S : 18S ratio ≥ 1.4).
Sample Quality Good Option: A260/280 ≥1.9 and a formaldehyde 1% agarose gel with the integrity of RNA judged upon staining with ethidium bromide. High quality eukaryotic RNA will show a 28S rRNA band at ~4.5 kb. This band should be twice the intensity of the 18S rRNA band at ~1.9 kb. Both kb determinations are relative to a RNA 6000 ladder [provided in the Agilent 2100 Bioanalzyer RNA 6000 kit]. High quality prokaryotic RNA will show a 23S band at twice the intensity of the 16S band; size is dependent on organism. The mRNA will appear as a smear from 0.5.12 kb. (Adapted from mRNA-Seq_Sample Prep 1004898_RevA Manual from Illumina, Inc.).

Sample Documentation Best Option: Provide pdf printout of bioanalyzer results.
Sample Documentation Good Option: Provide printout of A240–A320 spectral scan with ratio of 28S:18S band (or ratio of 23S:16S for RNA obtained from bacteria), A260/280 ratio and gel picture with clearly labeled ladder and sample(s).

Sample Quantity: 20ug ≥ 100 ng/uL unless arranged otherwise on Service Agreement.

mRNA Sample
Sample Quality Best Option: Ribosomal RNA contamination <10% determined using Agilent 2100 Bioanalzyer RNA6000 Pico Chip to check quality and quantity.
Sample Quality Good Option: A260/280 ≥1.9 and a gel picture of mRNA w/ clearly labeled ladder and sample(s). The mRNA should show NO ribosomal RNA bands.

Sample Documentation Best Option: Provide pdf printout of bioanalyzer reading results
Sample Documentation Good Option: Provide printout of A240–A320 spectral scan, A260/280 ratio and gel picture with clearly labeled ladder and sample(s).

Sample Quantity: 300 ng at ≥ 10ng/uL unless arranged otherwise on Service Agreement.

MicroRNA Sample
Sample Quality Best Option: Presence of two microRNA peaks between ~18–30 nt determined using a Agilent 2100 Bioanalzyer Small RNA kit.
Sample Quality Good Option: A260/280 ≥1.9 of the microRNA and bioanalzyer result or gel picture of the Total RNA from which it was extracted. See requirements for Total RNA Quality for specifications.

Sample Documentation Best Option: Provide pdf printout of bioanalyzer results.
Sample Documentation Good Option: Provide printout of microRNA 260/280 result or results of Total RNA bioanalzyer or gel with clearly labeled ladder and sample(s).

Sample Quantity: 500 ng at ≥ 15ng/uL unless arranged otherwise on Service Agreement.

Sequencing Types Definitions:
SR = Single End Read
PE = Paired End Read
SR—MP = Single End Read, Multiplexed
PE—MP = Paired End Read, Multiplexed