Overview

The amount of RNA that can be extracted from different biological or clinical samples varies greatly. For example, while a few micrograms of RNA could be easily purified from tissues and cells in excess amounts (such as from a few milligrams of tissue), many liquid biopsy samples may yield very low amounts of RNA. In fact, samples such as urine or plasma may yield 1 - 100 ng or less RNA per 100 µL of sample. Such a range of RNA quantity is often below the detection limit of most commonly used techniques for measuring RNA including nano-spectrophotometry and fluorescent nucleic acid stains. As a result, without properly determined RNA concentration, it becomes very difficult to normalize the starting quantity of RNA used in gene expression studies.

Norgen’s microRNA (cel-miR-39) Spike-In Kit offers a quantified synthetic RNA (cel-miR-39) for spike-in during RNA extraction procedures and subsequent normalization in RT-qPCR assays. The amount of cel-miR-39 RNA recovered after RNA extraction is directly correlated with the amount of total RNA recovered. After reverse transcription (such as with Norgen’s microScript Reverse Transcription system) of the sample RNA (with spike-in), the level of cel-miR-39 can be determined by subjecting the cDNA generated to quantitative PCR (qPCR) using fluorescent nucleic acid stains such as SYBR Green.  A cel-miR-39 specific primer is included in the kit. The level of expression of any target transcripts in different RNA samples can now be normalized to the cel-miR-39 transcript level using standard method such as ∆∆Ct relative quantification.

In addition, the cel-miR-39 RNA is compatible to library preparation methods (including ligation-based protocols) in Next Generation Sequencing (Small RNA-Seq) workflows. The cel-miR-39 RNA could be used for normalization as well as for tracking library construction efficiency.

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Supporting Data

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Figure 1. Accurate Quantity and High Quality cel-miR-39 Spike-In. Various quantities of the reconstituted cel-miR-39 RNA was used as spike-in during isolation of urinary RNA (1 mL) using Norgen's Urine Exosome RNA Isolation Kit (Cat. 47200). Isolated RNA was reverse transcribed using Norgen's microScript cDNA Synthesis Kit (Cat. 54410), followed by qPCR using Norgen's 2X PCR Master Mix (Cat. 28007), supplemented with SYBR Green I. The cel-miR-39 RNA was successfully detected in all samples, with the Ct values distributed according to the amount of RNA spiked-in.

Figure 2. Successful Incorporation of cel-miR-39 Spike-In into Small RNA-Seq Library. Small RNA-Seq Library was generated using either a competitor’s or Norgen's cel-miR-39 Spike-In RNA at two concentrations (High and Low). The library was generated using Illumina's TruSeq Small RNA Library Preparation Kit. The incorporated libraries were resolved on a PAGE gel. Only Norgen's cel-miR-39 RNA showed effective incorporation into small RNA-Seq library (Red Arrows). The competitor's cel-miR-39 RNA only yielded bands (Yellow Arrows) equivalent to the No Template Control (NTC) that are adapter dimers.

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