Targeted NGS for investigating the mitochondrial genome and heteroplasmy
Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants.…
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Total RNA Sample Preparation for RNA Analysis Studies
Sample Amount Required
The amount of total RNA required for library preparation depends on the profiling service requested. Please see our RNA sequencing service comparison chart for sample requirements. If possible, we ask that you try to send more than these.
Total RNA should be shipped in DEPC treated water at a concentration >100 ng/µl.
Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (0.5 ml or smaller PCR tubes can crack when frozen).
Total RNA Extraction
You can use any one of the commercially available column based RNA extraction kits that are specifically developed for total RNA extraction, from vendors such as Norgen Biotek, Qiagen or Ambion. If you have multiple samples, make sure to use only one type of extraction kit for all the samples of your project.
Many laboratories have obtained excellent results from total RNA samples extracted using Trizol methods. However, skills, experiences and sometimes sample types may become critical factors in obtaining consistently good sample qualities. According to our statistics, this method has an overall higher failure rate than column-based commercial methods, although the rate varies among different laboratories.
Perform a DNase treatment on the RNA sample.
Please transfer your sample to a 1.5ml microcentrifuge tube for shipment (smaller tubes can crack when frozen).
Note – We now offer an RNA extraction service for our US customers who would prefer to just send us cells or tissue samples.
Quality Control of RNA Sample
High quality results are dependent on high quality samples. There are several methods you can use to check the total RNA quality before shipping:
Measure the 260/280nm ration with a UV spectrophotometer. It should be in the range 1.8-2.0.
Run your sample on a Bioanalyzer, if you have access. The RNA Integrity Number (RIN) should > 8.
Alternatively, run your sample on a formaldehyde 1% agarose gel and stain with ethidium bromide. High quality RNA will show a 28S rRNA band at 4.5 kb that should be twice the intensity of the 18S rRNA band at 1.9 kb. Both kb determinations are relative to a RNA 6000 ladder. The mRNA will appear as a smear from 0.5–12 kb.