TaKaRa SMARTer pico RNA kit

The Takara SMARTer Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian kit is specifically designed for very low input total RNA samples. It also works with degraded total RNA.

We recommend opting for the SMARTer Stranded Total RNA-Seq Kit v3 – Pico Input Mammalian if you have small amounts of RNA (10 ng or less), or degraded samples for e.g. sourced from FFPE tissues. This kit is an updated version of the original SMARTer Stranded Total RNA-Seq Kit v2, which we previously offered, with the additional advantage of including Unique Molecular Identifiers (UMIs) – in sequencing read 2 – to mitigate any bias introduced as a result of PCR. We anticipate that this will also enable you to quantify transcripts more effectively – particularly to help identify true variants and rare mutations.

Please note that we will no longer be offering the v2 kit. Please get in touch in case of any specific concerns or queries.

  • This protocol is suited for:
    • Total RNA samples that are partially degraded (for e.g. sourced from FFPE tissue).
    • Small amounts of total RNA (eg. 10 ng or less; best performance for the 250 pg – 10 ng range and 10 ng and above for FFPE-sourced total RNA).
    • Samples that have a very low concentration (sorted cells, LCM).
    • Applications where depletion of cytosolic rRNA or human mitochondrial rRNA is suitable.
    • Applications where strand information is required.

Sample requirements

  • Sample type: Total RNA
  • Sample concentrations and amounts used for prep:
    • Recommended concentrations: 1.25 – 25 ng/μl.
      • When samples with concentration >1.25 ng/μl are provided, 10 ng will be used for library prep.
      • When samples with concentration <1.25 ng/μl are provided, all the material will be used for prep (up to 8μl).
    • For degraded RNA for e.g. sourced from FFPE, a minimum of 10 ng input is recommended for library prep.
  • Sample volume: min 12 μl (4 μl used for initial quality checks and 8 μl for library prep).
  • Sample quality: high quality (RIN > 7; DV200 ≥ 50%) or degraded (we do not recommend library prep of samples where DV200 < 30%).
  • Sample extraction method: we recommend using a column-based kit for extraction since residual phenol can interfere with prep.
    • The samples should be eluted in nuclease-free water.
    • Elution Buffers interfere with the library prep and should be strictly avoided.
  • DNase I treatment is required, preferably performed on-column during the extraction.

Evaluating Sample Quality

All samples we receive are assessed for quality and concentration “Reception Control”. However, we require our users to provide us with their own estimates to serve as a frame of reference. We recommend the following:

  • To measure RNA concentration
    • Fluorometric measurements (Qubit, Quant-it)
    • Do not use absorbance measurements (Nanodrop, spectrophotometer)
  • To assess RNA quality
    • Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation)
    • Do not use gel electrophoresis
  • To assess RNA quality for degraded and/or FFPE samples
    • Use capillary electrophoresis to determine the DV200

Please contact us if the quality and concentration of your sample(s) cannot be assessed using the recommended methods or if they are below the range of detection.

What we do with your samples

Once your samples arrive at NGI, we start by performing a reception control step where we measure the concentration, volume, and the DV200 of your samples.
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.

Library preparation

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 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 normalized, pooled and queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement. Please note that read 2 will be eight base-pairs shorter since the UMIs will be included in this read.

Expected results

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 depend on the individual 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.

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We are routinely running this method. Please visit the Order Portal to place an order.

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