Friday, July 20, 2012

How to read your RNA QC results

High quality RNA is needed for further processing of your samples. When we receive RNA for gene expression, QPCR, or miRNA, all samples are "QC'd" first. QC stands for quality control. How we do this is first OD samples either through Ribogreen or Nanodrop. Then depending on the amount we either run the samples on the a nano or pico chip. Once we finish our QC, we usually send you the OD results and Bioanalyzer results. We often get questions about what good RNA results look like, thus we thought we would simplify the explanation in three short steps:

1). You will receive a document that looks similar to the one below. This is an actual user's results so all identifying characteristics, such as name, have been blocked out. Your sample's gel picture should show two distinct bands representing the 18S and 28S ribosomal RNAs.

2). Most importantly is the electropherogram that shows fluorescence of the two 18S and 28S ribosomal RNA peaks. High quality RNA has this profile.
3). Agilent also provides a calculated number called the RNA Integrity Number (RIN). Although it can be a good indication of high quality RNA, it is sometimes unreliable. We would never rely solely on this number to tell us about the RNA's quality.

The combination of these three entities helps to give us the full picture of RNA quality. Also every project is considered individually. If you have any questions please check the About the Core page for contact information.

Tuesday, July 17, 2012

Information about RNAseq


In the Genomics Core Lab we define two different types of RNAseq
  • Transcriptome Analysis
  •  Transcriptome Discovery

For a Transcriptome analysis, we will generate an amount of data similar to an microarray data, between 20 and 30 million reads on a Paired-end Hiseq run (Paired end means that each fragment will be sequenced in both direction, 50bp from the reverse side, and 50bp from the forward side). The dynamic range will be similar as the one observed on an Affymetrix or an Illumina Gene Expression array.

Cost of transcriptome analysis : ~ $500/sample  (from total RNA to raw data)

In a Transcriptome discovery, you will get exon level information, you should be able to monitor expression of rare transcript and hopefully you should be able to identify known as well as new fusion events. We will generate between 80 and 100 million reads per sample, on a Hiseq Paired end 75bp/75bp run.

Cost of transcriptome discovery: ~$900/sample (from total RNA to raw data)


Last update
7/20/2012


20-30 M (6 samples/lane)
80-100 M( 2samples/lane)
2x50
$404
$844
2x75
$462
$1,019
2x100
$492
$1,108


Some frequently asked questions...

Amount of material needed?   
The standard protocol calls for 500ng of total RNA.
Less is doable, but would have to be treated on a case by case based on the amount.

What is the lowest amount of RNA I can submit?  
 We have processed samples that are below the threshold of detection of ribogreen or Agilent BioAnalyzer, coming from as low as 7 cells. We are actively working on a valid single cell protocol.

If your samples fall in this category, keep in mind that the amplifications always introduce bias in the data, no matter what method of  amplification we will use –PCR, IVT, Rolling amplification-. Another consequence is that instead of 70 to 80% of the data being informative, probably only 30 to 40% will be informative. On the bright side of things, 30 to 40% is better than 0% (which is what you would get if you don’t try!).

Will my samples be barcoded? 

Yes. For transcriptome analysis, we will put  up to 5 samples per hiseq 2000 lane. For transcriptome discovery, we will sequence 2 samples per lane. This difference is what is dictating the difference in cost.
Quality of RNA?
A RIN (RNA Integrity Number) of 8 is ideal, however, as for a microarray experiment, what is essential is homogeneity among a sample set .

Biological replicates
Absolutely YES. You will need some of these if you want to reduce the number of false positive targets.

Turnaround time? 
3 weeks at the minimum.  A typical break down of the time is described below:
Week 1: RNA QC, OD, library making
Week 2: preparation of the flow cell for sequencing (Monday)
Week 2: Tuesday : start the run (12 days)
Week 3; Sunday , end of the run
Week 4: Monday : data archiving, mapping, and quality control.
Week 4: Wednesday. Data ready for secondary analysis.

Additional cost? 
If you do not have the expertise to transform raw data into a gene expression matrix or fold change, you will need the assistance of the bioinformatics core.


Can a transcriptome discovery run also be used for transcriptome quantification?
Absolutely yes

Can we go back and get higher read depth with a library after a run, either for the same read length or the other read length?
Absolutely yes

 

Wednesday, July 11, 2012

DNA Bling

When you love what you do sometimes you want to wear it, on your ears. Or maybe around your neck and your wrists. The other day our director came by the lab and as she was talking I was drawn to her earrings because they were in the shape of DNA!-the beloved substance of life that we work with everyday.



Inspired by her earrings I went out on a search for the best "DNA Bling" across the web. Here are my top pics:

1. "It's all in your genes" bracelet is very dainty piece and the creator has made each nucleotide base a different stone: amethyst- adenine, tourmaline- thymine, citrine- cytosine, garnet- guanine.



2. DNA Wire Spring Earrings: For  the more bold, and funky earring wearer, we have these triangular wire earrings with a helix at the bottom.



3. DNA Genetics Cabochon Brass Ring: I had to throw in a ring in this round up. This ring is a very nice, understated brass ring with what looks like a karyotype image inside.

4. DNA Base Pair Friendship Bracelet:A personal favorite of mine, this has got to be the cutest thing. Lab buddies can display their friendship in terms of base pairing. A and T are also available.

5. Etched Copper DNA Double Helix Bracelet: For the bohemian budding scientist who wants to evoke more earth tones this bracelet is definitely for you!

6. Moma DNA Bracelet: For those who want to rock "DNA Bling" without looking like you are doing just that.


7. Frank Gehry® Orchid drop pendant” While this pendant is not originally advertised to be a DNA inspired necklace, it looks like it and gives us an excuse to throw in something from the coveted Tiffanys. 


8.  DNA Cuff Links: Of course we could not forget the dapper "fellas" who love science.



I hope you enjoyed this round-up. Who knew there were  people paying homage to DNA through the art of jewelry making?  I'm now on a mad hunt for DNA inspired home accessories. Post to come soon!   
Please comment and check out the links above. If you have seen any other cool jewelry DNA pieces not featured, please send them our way!

Monday, July 9, 2012

Article To Watch: Options in high through-put sequencing

Ion Torrent Machine
Roche 454 Junior
Illumina Miseq
With the recent publishing of the Nature Biotechnology article, Performance comparison of benchtop high-throughput sequencing platforms, showcasing the differences in three common "bench-top" sequencers, The 454 GS Junior (Roche), The Miseq (Illumina), and the Ion Torrent PGM (Life Technologies), we thought we would highlight the article seeing that our core offers services using all three platforms. The bottom line of the article highlights that choosing between these three platforms is a multi-pronged decision in that it is ultimately based on the user's needs. For more information on which platform would best fit your sequencing needs, check out the article and then feel free to email us at genomics@mskcc.org .




Nicholas J Loman, et al. (22 April 2012). Performance comparison of benchtop high-throughput sequencing platforms [Online Article].Retrieved from http://www.nature.com/nbt/journal/v30/n5/full/nbt.2198.html#corres-auth.