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, eg. from FFPE tissues.
- 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.
- Recommended concentrations: 1.25 – 25 ng/μl.
- 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.
- Lower than recommended inputs will result in the generation of unmapped reads, PCR artifacts and/or low-quality reads.
Bioinformatics
FAQs
I have very low amounts (ng to pg) of total RNA, could those be used for a sequencing project with NGI?
Yes, NGI offers users the TaKaRa SMARTer total RNA-seq v3 Pico Input Mammalian kit – a library preparation method designed for low amounts or degraded input RNA. Details on our sample requirements are listed here.
This method is also suitable for investigating non-polyadenylated transcripts such as lncRNA and mitochondrial RNA from human cells, because it applies random priming to capture all transcripts.
Overall, low starting amounts of RNA risk of yielding low complexity data.
What kind of input material can I supply to NGI for library preparation using the TaKaRa SMARTer total RNA-seq v3 Pico Input Mammalian kit?
The TaKaRa SMARTer total RNA-seq v3 – Pico Input Mammalian kit is designed for low input and/or degraded RNA obtained from mammalian cells, FFPE samples and liquid biopsies.
RNA samples must be eluted in nuclease-free water, be devoid of phenol, and be treated with DNAse to rid them of any contaminating DNA since it can act as a template for reverse transcriptase and contaminate the final cDNA library. Detailed sample requirements for can be found here.
While the kit recommends 250 pg – 10 ng of total RNA as an input, we have successfully prepared libraries with lower inputs from non-FFPE sources (for example <1 ng) and a maximum amount of 50 ng. However, we cannot provide any guarantees of success with input amounts of less that 10 ng. Please contact us at support@ngisweden.se if you have any questions about suitability of your samples.
We require at least 10 ng of total RNA isolated from FFPE sources for library preparation with this method.
What amounts of RNA would you require for samples isolated from FFPE material?
We ask for a minimum of 10 ng of total RNA isolated from FFPE samples. Please consult our sample requirements for details on concentrations and quality of RNA samples that we accept for this method of library preparation.
Which method should I use to isolate RNA from my samples?
We recommend a column-based method of purification that includes a step with DNase digestion, since any contaminating DNA can act as a template for reverse transcriptase and contaminate the final cDNA library. This is especially critical for RNA that is isolated from FFPE and PFA fixed samples because they can contain large amounts of ss-DNA.
For tips on the different kit types that one could use, please refer to the manufacturer’s guidelines.
Which buffer should I use to elute my RNA samples?
Please elute your RNA in nuclease-free water. Also note that it is essential that samples are devoid of even traces of phenol.
Moreover, RNA stored in RNAlater need to be purified to be made compatible to this method of library preparation.
My samples were isolated using phenol-chloroform, are they suitable for library preparation using the TaKaRa SMARTer total RNA-seq v2 Pico Input Mammalian kit?
Unfortunately not; residual phenol can interfere with the enzymes used during the various steps. However, if you feel assured that you have removed residual phenol from your samples, please feel free to contact us at support@ngisweden.se to discuss your options.
Do my RNA fragments need to be of a specific length to be suitable for library preparation?
The longer and the more intact the RNA, the better the quality of the data. The manufacturer recommends lengths > 100 nt; one of the advantages of this method is its suitability for long transcripts due to random priming. For more details, please consult our tech note.
NGI does not fragment the RNA unless explicitly asked for by the user. If a prior project with included a fragmentation step and you would like for the same protocol to be followed, please let us know while placing your order.
Do I need to deplete my RNA of ribosomal RNA prior to delivering my samples to NGI?
No, submitted samples do not need to be depleted of rRNA prior to delivery. The TaKaRa SMARTer total RNA-seq v2 Pico Input Mammalian kit uses a proprietary method – ZapR – aided by probes that selectively target 18S and 28S rRNA and m12S and m16S rRNA from mammalian cytosolic and human mitochondrial ribosomes respectively, making this kit suitable only for mammalian samples. This step is carried out after cDNA synthesis.
non-rRNA RNAs i.e. mRNAs, mtRNA, lncRNA etc are then enriched for after a second round of PCR.
While in older versions of the kit, a fraction of rRNA was retained even after treatment with ZapR, this is no longer the case for v2 and v3 protocols.
I have very low amounts of RNA from a non-mammalian eukaryote. Could those be used to prepare libraries using this method?
The TaKaRa SMARTer total RNA-seq v3 Pico Input Mammalian kit is tailored for mammalian-derived samples. This is because the ZapR probes that are used to degrade the rRNA are specific to rRNA of mammalian cytosolic and human mitochondrial ribosomes.
However, we have prepared libraries from eukaryotic samples of non-mammalian origins with some degrees of success. Please note that we cannot provide any guarantees of success at attempts to prepare libraries or the data that results from libraries that are sequenced. Feel free to contact us if you have any questions or would like to try out this method on non-mammalian samples.
What information do I need to provide NGI with when submitting samples for this prep?
NGI provides users with a template which is to be filled out with sample information prior to sample delivery.
For library preparation of RNA samples using the TaKaRa SMARTer total RNA-seq v3 Pico Input Mammalian kit, we ask users to provide us with their estimates of concentration, volume, and DV200 values (preferably over 50%, but a minimum of 30%). Specific details are listed in our sample requirements for this method.
We understand that picogram amounts of RNA are hard to estimate. Please contact us to discuss your options if you have used a column-based method of purifying RNA and are confident of having RNA in your sample!
I was notified that my samples failed reception control. What are my options?
Given the uncertain quality and concentration of samples, we are unable to provide any guarantees that our attempts at library preparation will be successful. We also do not offer re-attempts at library preparation with samples that fail our requirements.
Low inputs of RNA (<10 ng) and highly degraded input material (DV200 < 30%) can fail to yield libraries that would generate meaningful data after sequencing. However, should you choose to proceed with said samples, we will charge you the full cost of library preparation and sequencing. We are not liable for low-complexity data that result from low input or degraded samples.
If we encounter technical errors during library preparation, you will be provided with the option of delivering more samples to us. However, please note that if a majority of your samples pass QC and a few don’t, we will not ask you for replacement samples since that will be attributed to the sample(s) itself.
Which sequencing instrument would NGI use to sequence the libraries made using this method of prep? How much data would I get?
Sequencing depths depend on the number of read-pairs you require for your specific scientific question. We recommend that you consult the literature for examples that could help determine the approximate number of read-pairs that you would need.
Please bear in mind that libraries prepared using this method could have a 5’ bias due to the random primer method used to generate them. Moreover, low starting amounts of RNA will result in low complexity data with higher rates of duplication; greater sequencing depths will not yield more complex data.
For a quick guide to sequencing depths, reads, and approximate costs of sequencing on the numerous flow cell types we offer, please refer to our price examples page under the tab “Illumina sequencing”. You are also welcome to request a meeting with us through our order portal or by email at support@ngisweden.se.
I would like to extract RNA from cells, are there any lysis conditions that you would recommend using?
We recommend incubating cells for 3 minutes at 98°C to rupture them prior to RNA isolation.
Can I detect mitochondrial transcripts with this method?
Yes, the TaKaRa SMARTer total RNA-seq v2 Pico Input Mammalian kit is suitable for detecting mitochondrial transcripts from total RNA since it uses a random priming method to select RNA.
I provided pg amounts of RNA to NGI for sequencing. Can I use the sequencing data that I get for DGE?
Our experience is that less than 1 ng of starting amounts can increase the risk of duplicates. In general, low concentrations and quality present lower complexity and therefore higher rates of duplications in the cDNA libraries.
Please take this into account when setting up a project with low inputs of RNA and contact us at support@ngisweden.se to discuss any questions you might have.
Last Updated: 3rd April 2024