| Sign In | Quick Order CART (0)
  • Ordering

    Cat. No.
    Size
    List Price*
    QTY
    BIO-52076
    10 Preps
    $65.00
    +
    BIO-52077
    50 preps
    $286.00
    +
    *To check your pricing please Login or contact us

    Description

    Highly efficient purification of total RNA by combining the stringency of either guanidinium thiocyanate or guanidinium hydrochloride with the speed and purity of silica-membrane column purification.

    Product Highlights

    RNA was isolated from 20 mg freeze-dried budding leaves of A. thaliana using ISOLATE II RNA Plant Kit. The extracted RNA was diluted in a 2-fold serial dilution (lanes 1-7) and PCR was performed using MyTaq One-Step RT-PCR Kit. The results illustrate the quality of the RNA obtained, as it can be used for very sensitive cDNA synthesis and PCR without further purification.

    Product Description

    The ISOLATE II RNA Plant Kit provides a simple, efficient column-based method for the isolation of total RNA from a wide variety of plant materials, including leaves, bark, roots and fruits, without the need for hazardous reagents such as phenol.

    By combining the stringency of guanidinium thiocyanate lysis with the speed and ease-of-use of silica-membrane purification, the ISOLATE II RNA Plant Kit provides a fast method for the purification of high-quality total RNA from most plant cells, including plant tissues, leaves, bark, roots and fruits.

    Some plant tissues such as maize endosperm and the mycelia of filamentous fungi do not lyse well in guanidinium thiocyanate and can solidify, resulting in poor yields and so guanidinium hydrochloride lysis buffer is also provided as an alternative. Residual amounts of contaminating DNA are selectively removed using a column, however for ultrasensitive applications, remaining DNA can be removed using RNase-free DNase I that is supplied with the kit. 

    The ISOLATE II RNA Plant Kit has been designed to deliver optimal performance in RT-qPCR in conjunction with either the SensiFAST cDNA Synthesis Kit and SensiFAST Real-Time PCR Kits, or the SensiFAST One-Step Real-Time RT-PCR Kits. Additionally, the ISOLATE II RNA Plant Kit can be used to purify samples prior to RT-PCR amplification using the Tetro cDNA Synthesis Kit and any enzyme from the Bioline PCR portfolio, including MyTaq DNA Polymerase.

    Applications

    • RT-qPCR
    • End-point RT-PCR
    • Northern, dot and slot blotting
    • Array analysis
    • Poly A+ RNA selection
    • RNA-Seq
    Main
    Fast purification of high-quality total RNA from a wide variety of plant tissues, including leaves, bark, roots and fruits.

    Nucleic Acid Isolation Guide

    Download the ISOLATE II Guide with detailed product descriptions and performance data to help you choose the best product for your research

    Specification

    Components

    Reagent

    10 Preps

    50 Preps

    ISOLATE II Filters

    10

    50

    ISOLATE II RNA Mini Columns

    10

    50

    Collection Tubes (2 mL)

    30

    150

    Collection Tubes (1.5 mL)

    10

    50

    Lysis Buffer RLY

    10 mL

    25 mL

    Lysis Buffer RLS

    10 mL

    25 mL

    Wash Buffer RW1

    15 mL

    15 mL

    Wash Buffer RW2

    6 mL

    12 mL

    Membrane Desalting Buffer MEM

    10 mL 

    25 mL

    Reaction Buffer for DNase I RDN

    7 mL

    7 mL

    DNase I, RNase-free (lyophilized)

    1 vial

    1 vial 

    RNase-free water

    13 mL

    13 mL

    Bench Protocol Sheet

    1

    1

    Storage & Stability

    All components should be stored dry and at room temperature.

    When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.

    Shipping conditions

    Ambient temperature.



    FAQs

    The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.

    Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.

    Several possibilities exist. If you are using our column based extraction kit like the ISOLATE II RNA Mini kit it would be necessary to decrease the sample amount of such samples or to increase the amount of the lysis buffer RLY to prevent clogging of the columns. Another possibility would be a pre-extraction with TRIsure (BIO-38032) and to clean up the RNA containing aqueous phase with the column based ISOLATE II RNA kits. You should follow the TRIsure protocol up to the phase separation step. Then mix the aqueous phase with a volume of ethanol and load it onto the column. From there you should proceed with the regular ISOLATE II RNA Mini kit protocol.

    These samples are treated as usual, just remove the RNAlater solution.

    Agarose gel analysis of RNA samples can be both valuable and misleading. The pattern of bands on the gel can not only be indicative of the quality of the sample, but equally it can also only indicate that the gel tank, buffer or agarose is contaminated with RNase. A safer measure is to use a Bioanalyzer and look at the RIN value (RNA Integrity Number), which should be as close to 10 as possible, indicating that the RNA is not degraded. A spectrophotometer (ideally a microfluidic one) can also be used to determine the ratio of A260 to A280 for purity determination.

    All these methods give an idea of the quality of total RNA which tends to be dominated with rRNA and tRNA. It is not uncommon for an agarose gel to show an abundance of 18S and 23S rRNA but on analysis for there to be little of the transcript of interest. Ideally RNA quality control should include an assessment of the presence of common transcripts (from reference genes, such as GAPDH) using RT-PCR or RT-qPCR.

    If the yield of RNA is low, it is best to first check that all the solutions used and the equipment employed is largely free of RNase. Solutions should be prepared with the highest quality reagents available, preferably ones that have a certificate of analysis to show that they are free of DNase and RNase. If there is confidence that RNase contamination is at a minimum, then it may be worth ensuring that the sample is properly homogenised before lysis and to check a sample of the lysed material under a light microscope to ensure that all the cells are disrupted.

    RNA samples that have been contaminated with RNase (either during processing or those that have been sent from another lab) can be purified using the ISOLATE II RNA Micro Kit.



    We recommend the ISOLATE II Biofluids RNA kit for the extraction of cell free RNA. The kit isolates all sizes of RNA and is compatible with very limited, or challenging sample sources containing low RNA content.

    We recommend our ISOLATE II Biofluids RNA, miRNA and Plant miRNA kits. The miRNA kits are able to capture all small RNA species (<200 nt) from plant cells and tissues and to purify high quality large RNA (>200 nt) from the same sample in parallel. The ISOLATE II miRNA Kits have been developed to overcome the bias observed with small RNA isolation using phenol-based techniques, due to its proprietary small RNA separation and enrichment technology, as well as highly optimized chemistry. The Biofluids RNA kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA down to small RNAs such as miRNA and siRNA. The ISOLATE II Biofluids RNA Kit is compatible with very limited, or challenging sample sources containing low RNA content.



    The lysis buffer RLY and RLS contain guanidinium thiocyanate and guanidinium hydrochloride, respectively. Lysis buffer RLY works in most cases and is recommended for the lysis of most plant materials due to the stronger denaturation properties. However, some plant tissues or fungi solidify in RLY and RNA purification cannot proceed, buffer RLS should be used instead.