The right answers to frequently asked questions
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Which INVIEW Transcriptome package do you recommend for which application?
This depends on many factors, such as tissue type, sample quality, development status, existing references, etc. Please refer to the table shown under Product Specifications to find your optimum InView™ Transcriptome solution. The estimated read numbers shown there refer to human samples. Alternatively, please contact us to discuss your project. In some cases it might be advisable to set up a trial to define how many reads will be required for your specific aim or organism.
The INVIEW Transcriptome Explore package gives you an overview of the expressed genes and is well suited to detecting differences between samples.
INVIEW Transcriptome Discover uses a strand-specific RNA library combined with Illumina’s 150 bp paired-end sequencing and is therefore recommended for the detection of differentially expressed genes, rare and novel transcripts and for discovering splice variants. In addition, the information about the transcript’s orientation allows for a more precise determination of structure and gene expression.
RNA-Seq of bacterial samples most often requires less reads since no splice variants are present. The INVIEW Transcriptome Bacteria is a cost-alternative for all projects interested in the gene expression profile of Bacteria.
The INVIEW Transcriptome Ultra Low is specially designed for all projects with only a tiny amount of starting material.
Do I need a genomic reference sequence?
INVIEW Transcriptome Discover:
Not necessarily. However, if you have a reference, please refer to the recommendations below.
INVIEW Transcriptome Explore / Bacteria / Ultra Low:
A clearly defined Ensembl name (e.g., GRCh37 or Rnor_5.0) for the annotated genomic reference sequence has to be provided prior to project start. Alternatively, the genome sequence can be provided in Fasta (along with the corresponding annotation in Gene transfer format (gtf)) or in GenBank format (including annotation). For projects without a reference genome, de novo transcriptome sequencing based on PacBio RS II technology can be added.
Which starting material will be accepted?
Total RNA from tissues, cells or body fluids from eukaryotic or prokaryotic organisms can be used to prepare libraries for sequencing. In addition to RNA isolated from fresh tissue, RNA from FFPE samples can be analysed. RNA isolation can be offered as an additional service. For starting material from other sources please contact us.
INVIEW Transcriptome Ultra Low:
Please note that we cannot accept cells in cell culture media for the INVIEW Transcriptome Ultra Low package.
How much starting material is required?
For information on the quantity and concentration of RNA required for specific library preparation methods, please refer to your quote or contact us. In general, we require 150 ng of high-quality RNA (up to 25 µl / optimal concentration 6 - 50 ng/µl (absolute maximum concentration 200 ng/µl)) with an OD 260/280 of 1.8-2.0 and an OD 260/230 of 2.0-2.2 for optimum results. It might be possible to start with less material, but this depends a great deal on the quality of the material and the requested library.
The quality and quantity of the provided starting material are crucial for the success of sample preparation. The use of too little, degraded, contaminated or otherwise damaged starting material can result in a low yield or sample preparation failure and impair the quality and quantity of sequencing results.
INVIEW Transcriptome Ultra Low:
Minimum 0.15 ng/µl (in min. 10 µl); RIN > 6.5
Can RNA isolation be offered?
Eurofins Genomics offers RNA isolation as an additional service. We have successfully optimised protocols for extraction of high-quality RNA from several sample types and starting materials. Please refer to Product Specifications for more information on RNA isolation or consult us directly to discuss possible sample preparation methods for your individual requirements.
Which starting materials and aims are best suited for performing ribosomal RNA depletion?
a) For eukaryotes: rRNA depletion is recommended for partially degraded RNA or for when sequencing RNA with no poly-A tail.
b) For prokaryotes we recommend rRNA depletion in general, because poly-(A) enrichment of prokaryotic transcripts is not feasible.
You can order rRNA depletion from Eurofins Genomics. Or, alternatively, you can provide us with RNA that has already been rRNA depleted / mRNA enriched as starting material (20 ng per sample (up to 15 µl / concentration >1,4 ng/µl)). Please see Product Specifications for more information.
How efficient is ribosomal RNA depletion?
The efficiency of removal is determined by the organism, the composition and the quality of your sample RNA. If the RNA is severely degraded, ribodepletion might be less efficient.
Should I send my starting material on dry ice or would room temperature be sufficient?
RNA must be shipped on dry ice, unless the RNA is precipitated in ethanol. Tissues / cell cultures must be flash frozen in liquid nitrogen or dry ice and have to be shipped on dry ice. Alternatively, fresh material can be stabilised in “RNAlater” (e.g., Ambion, SIGMA or QIAGEN) and be sent at room temperature. Please verify in advance that the couriers you are using accept dry-ice shipments.
What should I do if my sample fails the entry QC?
If the amount, concentration or quality of the starting material does not meet requirements for further processing, we will contact you to discuss how to proceed further. If possible, Eurofins Genomics will recommend additional pre-processing steps in order to optimise sample quality.
Where and in what form can I obtain my results?
All raw data as well as the analysed data can be downloaded via your secure online account.
What kind of BioIT do you offer?
Affordable high-quality bioinformatics analysis, including data filtering and QC, is optionally available for each package.
Where should I send my samples?
Eurofins Genomics Europe Sequencing GmbH