TaKaRa SMARTer pico RNA kit
The Takara SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian kit is specifically designed for very low input total RNA samples. It also works with degraded total RNA.
The SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian is what we’d recommend if you have small amounts of RNA, or degraded samples. Please check the sample requirements and expected results below.
- You would want to use this protocol when:
- Samples are partially degraded, or low-quality (FFPE)
- You have a small amount of total RNA (eg. 10 ng or less)
- Samples have a very low concentration (sorted cells, LCM)
- Depletion of rRNA or mtRNA is suitable
- Strand information is required
- Sample type: Total RNA
- Sample amount:
- Recommended: 1.25 ng/μl
- If working with degraded RNA, a minimum of 10ng input is recommended for library prep
- Sample volume: min 12 μl (4 μl used for Reception Control and 8 μl for library prep)
- Sample quality: high quality or degraded (DV200 ≥ 30%)
- Sample extraction method: we recommend using a column-based kit without Phenol.
- The samples should be resuspended in nuclease-free water
- Elution Buffers interfere with the library prep
- DNase I treatment is required, preferably performed on-column during the extraction
How to evaluate the sample quality
Even if we perform a Reception Control on all samples, we require our users to asses both total RNA quality and concentration. To accomplish this we recommend using:
- RNA concentration
- Fluorometric measurements (Qubit, Quant-it)
- Do not use absorbance measurements (Nanodrop, spectrophotometer)
- RNA quality
- Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation)
- Do not use gel electrophoresis
- RNA quality for degraded and/or FFPE samples
- Use capillary electrophoresis to determine DV200
If your sample can not be assessed using the recommended methods or if it is below the range of detection, please contact us. We may still be able to generate a library from it.
What we do with your samples
Once your samples arrive at NGI, we start by performing a reception control step in which we make sure the sample meets our requirements.
If the samples fail this quality control step, we will contact you to discuss possible options.
If the samples pass reception control, we will inform you and the samples will be queued for library prep.
The protocol is ligation-free and thus preserves strand-of-origin information. Random priming allows the generation of cDNA from all RNA fragments in the sample, including rRNA and degraded mRNA.
The Reverse Transcriptase used adds additional nucleotides when it reaches the 5′ end. Those extra nucleotides are used for the synthesis of the second strand of the cDNA.
The next step is a short round of PCR amplification which adds full- length Illumina adapters, including barcodes.
The cDNA originating from rRNA is then cleaved by ZapR enzyme in the presence of mammalian-specific R-Probes. This process leaves the library with only fragments from non-rRNA molecules. These fragments are enriched via a second round of PCR amplification.
We have simplified the Library Preparation by skipping fragmentation and fixing the number of PCR cycles regardless of the RNA quality or input amount.
The simplified protocol version has been tested and the results are can be found in this Technote.
Library QC and sequencing
In this step, we evaluate the library yield and determine their size distribution. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are normalised, pooled and queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.
In general, we have been successfully generating libraries that provide enough good quality reads for differential gene expression analyses. However, the sequencing results from each library prep will depend on the characteristics of the sample:
- A high percentage of PCR duplicates (40% or more) when:
- The sample input is <10 ng
- The sample DV200 is <50%
- The sample diversity is intrinsically low
- The yields of the library prep could be insufficient when the input is <1 ng:
- When the library concentration is too low, generating an even pooling is more difficult, so you could expect to have uneven reads yields.