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[DESCRIPTION]
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<p>The ISOLATE II Biofluids RNA Kit is specially developed for the rapid phenol-free isolation of high quality total RNA from biofluids and viruses.</p>
<p> </p>
<p>The following protocol is two part. The first part provides a procedure for the removal of genomic DNA applies to all the different types of lysate. Ensure you use the correct column: the Genomic DNA Removal Column has a <strong>blue</strong> ring.</p>
<p> </p>
<p>The second part is the protocol for the purification of total RNA apply to all the different types of lysate. Ensure you use the correct column: the ISOLATE II RNA Column has a <strong>black</strong> ring.</p>
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[BEFORE_START]
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<p>Ensure Wash Buffer W1 is prepared (see guidelines).<a href="http://www.bioline.com" target="_blank">www.bioline.com</a></p>
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[GUIDELINES]
<p><strong>KIT CONTENTS</strong></p>
<p> </p>
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<p>COMPONENT</p>
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<p>50 Preps</p>
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<p>ISOLATE II RNA Columns (black)</p>
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<p>50</p>
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<p>ISOLATE II Genomic DNA Removal Columns (blue)</p>
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<p>50</p>
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<p>Collection Tubes (2 mL)</p>
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<p>100</p>
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<p>Elution Tubes (1.7 mL)</p>
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<p>50</p>
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<p>Lysis Buffer RX*</p>
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<p>40 mL</p>
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<p>Wash Buffer W1† (concentrate)</p>
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<p>38 mL</p>
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<p>DNase I Solution (RNase-free)</p>
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<p>0.8 mL</p>
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<p>DNase I Reaction Buffer DRB</p>
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<p>6 mL</p>
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<p>RNA Elution Buffer</p>
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<p>6 mL</p>
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<p>Product Manual</p>
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<p>1</p>
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<p>Bench-Top Protocol</p>
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<p>1</p>
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<p>*Contains a guanidine salt. Not compatible with disinfectants containing bleach or acidic solutions. </p>
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<p>†Before use, add 90 mL of 96-100% ethanol and mark wash buffer bottle label.</p>
<p> </p>
<p><strong>DESCRIPTION</strong></p>
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<p>The ISOLATE II Biofluids RNA Kit allows convenient processing of multiple samples in 20 minutes without the use of laborious methods such as CsCl ultracentrifugation or handling of toxic chemicals such as phenol/chloroform.</p>
<p> </p>
<p>The ISOLATE II Biofluids RNA Kit is specially developed for the rapid phenol-free isolation of high quality total RNA from biofluids and viruses. Total RNA can be purified from blood, plasma, serum and other types of biofluids such as saliva, urine, semen and cerebrospinal fluid (CSF). Viral RNA can be isolated from the same sample sources, as well as cultured cells and tissue. The kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA (rRNA) down to small RNAs such as microRNA (miRNA) and short interfering RNA (siRNA).</p>
<p> </p>
<p>Isolation is based on a fast spin column format using a novel RNA affinity resin as the separation matrix so that the small RNAs are preferentially purified from other cellular components. This kit does not require the use of phenol or chloroform.</p>
<p> </p>
<p>Samples are first lysed in the presence of guanidinium thiocyanate, a chaotropic salt which immediately deactivates endogenous RNases to ensure purification of intact RNA. DNA is then captured on a Genomic DNA Removal Column to remove contaminating genomic DNA. Ethanol is added to the flow-through and then applied to an RNA Column. The bound total RNA is washed to remove any impurities and eluted. The RNA is of the highest quality and integrity and is ready for use in various applications, including:</p>
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<p>Real-Time PCR (qPCR)</p>
</li>
<li>
<p>Reverse transcriptase PCR (RT-PCR)</p>
</li>
<li>
<p>cDNA synthesis</p>
</li>
<li>
<p>Next generation sequencing</p>
</li>
<li>
<p>Northern blotting</p>
</li>
<li>
<p>Microarray analysis</p>
</li>
</ul>
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<p>Please read this manual carefully to familiarize yourself with the ISOLATE II Biofluids RNA protocol before starting (also available on <a href="http://www.bioline.com" target="_blank">www.bioline.com</a>). More experienced users can refer to the Bench-Top Protocol for quick referencing during the procedure.</p>
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<p><strong>STORAGE</strong></p>
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<p>Store DNase I at -20°C upon arrival. All other components should be stored at room temperature (18-25°C). Storage at lower temperatures may cause precipitation of salts.</p>
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<p><strong>SAFETY INFORMATION</strong></p>
<p> </p>
<p>When working with chemicals, always wear a suitable lab coat, gloves and safety glasses.</p>
<p> </p>
<p>Lysis Buffer RX contains guanidinium thiocyanate. This chemical is harmful in liquid form when in contact with skin or ingested. If the solution is allowed to dry, the powder is harmful if inhaled.</p>
<p> </p>
<p><strong>CAUTION:</strong> Do not add bleach directly to solutions or sample preparation waste containing guanidinium salts. Reactive compounds and toxic gases can form. In the case of spillage, clean the affected area with a suitable laboratory detergent and water.</p>
<p> </p>
<p>For detailed information, please consult the material data safety sheet (MSDS) available on<br />our website at www.bioline.com.</p>
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<p><strong>PRODUCT SPECIFICATIONS</strong></p>
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<p>The ISOLATE II Biofluids RNA Kit is specially designed for the rapid isolation of total RNA including small RNAs (&lt;200 nt). The kit is compatible with biofluids such as blood, plasma, serum, saliva, urine, semen and CSF. Viral RNA can also be extracted from the same sample sources. The preparation time is approximately 20 minutes for 10 preps. The isolated RNA is of high-purity (A<sub>260</sub>/A<sub>280</sub> ratio: &gt;1.9) and high integrity (RIN&gt;9) for high quality samples (see below) and is ready for use in various downstream applications.</p>
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<p><strong>ISOLATE II BIOFLUIDS RNA COLUMN SPECIFICATIONS</strong></p>
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<p>Max. binding capacity</p>
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<p>50 μg RNA</p>
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<p>Max. column loading volume</p>
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<p>650 μL</p>
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<p>RNA size distribution</p>
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<p>All sizes, including small RNA &lt;200 nucleotides</p>
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<p>A260/A280 ratio*</p>
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<p>1.9-2.1</p>
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<p>Typical RIN (RNA integrity number) †</p>
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<p>&gt;9</p>
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<p>Max. amount of starting material:</p>
<p>Biofluid (Blood/Saliva/Semen/Urine/CSF)</p>
<p>Plasma/Serum<br />Viruses</p>
<p> </p>
<p> </p>
<p> </p>
<p>Biofluid in Cotton Swab</p>
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<p>100 μL</p>
<p>200 μL<br />3 x 10<sup>6</sup> cells<br />~20 mg mammalian tissue<br />100 μL viral suspension or blood</p>
<p>One swab</p>
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<p> </p>
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<p><em>*Typically, the A<sub>260</sub>/A<sub>280</sub> ratio exceeds 1.9, indicating excellent RNA purity.</em></p>
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<p><em>† Agilent 2100 Bioanalyzer (RNA 6000 assay). RNA integrity is highly dependent on sample quality.</em></p>
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<p>The following components are also included in the kit:</p>
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<li>Genomic DNA Removal Column for removing contaminating genomic DNA.</li>
<li>DNase I (RNase-free) for eliminating genomic DNA contamination by on-column digestion or by digestion in solution (for the most sensitive applications).</li>
</ul>
<p><strong>Biofluid RNA Isolation</strong></p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/g39c2w6.png" data-src="https://s3.amazonaws.com/pr-journal/g39c2w6.png" data-ofn="Screen Shot 2016-12-06 at 3.47.31 PM.png" /></p>
<p><strong>EQUIPMENT AND REAGENTS TO BE SUPPLIED BY USER</strong></p>
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<p>When working with chemicals, always wear a suitable lab coat, protective goggles and disposable gloves. Please consult the relevant MSDS from the product supplier for further information. </p>
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<p>The following may be supplied by the user:</p>
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<p>ß-mercaptoethanol (ß-ME)* (Optional for Lysis Buffer RX)</p>
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<li>
<p>96-100% ethanol† (for Wash Buffer W1)</p>
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<li>
<p>Equipment for sample disruption and homogenization (see section 7.2). One or more of</p>
<p>the following are required depending on chosen method:</p>
</li>
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<p>           o PBS (RNase-free) and trypsin<br />           o Needle and syringe (both RNase free) o Mortar and pestle</p>
<p>           o Rotor-stator homogenizer</p>
<p>           o Liquid nitrogen</p>
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<li>
<p>Molecular biology grade water</p>
</li>
<li>
<p>RNase-free microcentrifuge tubes (1.5 mL)</p>
</li>
<li>
<p>Sterile RNase-free tips</p>
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<p>Sterile cotton swabs (for Nasal/Throat Swab Protocol)</p>
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<li>
<p>MS2 RNA (for Plasma/Serum Protocol)</p>
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<p>Benchtop microcentrifuge (capable of 14,000 x g)</p>
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<p style="display: inline !important;"><em>*TCEP is also a suitable reducing agent instead of ß-ME.</em><br /><em>† Molecular biology grade ethanol is recommended. Do not use denatured alcohol which contains unwanted additives such as methanol and acetone.</em></p>
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<div class="column"><strong>IMPORTANT NOTES</strong></div>
<div class="column">The protocol steps are homogenization and lysis from different starting materials, genomic DNA removal, and purification of total RNA. The DNA removal and RNA purification protocols apply to all starting materials. Optional DNase I treatment protocols are provided.</div>
<div class="column"><br />• <a href="https://www.protocols.io/view/isolate-ii-biofluids-rna-kit-f5kbq4w" target="_blank">The collection</a> contains the lysate preparation protocols for various starting materials; ensure<br />you follow the specific protocol for your sample.<br />• The instructions for removing genomic DNA  and purification of total RNA from all types of lysate are described in <a href="https://www.protocols.io/view/total-rna-purification-from-all-types-of-lysate-f48bqzw" target="_blank">this protocol</a>.<br />• For isolating RNA from plasma/serum samples, follow <a href="https://www.protocols.io/view/support-protocol-total-rna-purification-from-plasm-f5hbq36" target="_blank">this dedicated protocol</a>.<br />• <a href="https://www.protocols.io/view/appendix-b-optional-dnase-i-treatment-of-purified-f5jbq4n" target="_blank">This</a> contains the optional protocols for additional DNase I treatment.</div>
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<div class="column">The ISOLATE II Biofluids RNA purification procedures can be performed at room temperature. Handle the eluted RNA carefully to avoid contamination by RNases, often found on labware, fingerprints and dust. For optimal RNA stability, keep RNA frozen at -20°C for short-term or -80°C for long-term storage. When working with RNA samples in downstream applications, keep the RNA solution on ice.
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<p> </p>
<p>Two types of spin columns are provided with this kit: the ISOLATE II Genomic DNA Removal Column (blue ring) and the ISOLATE II RNA Column (black ring). Ensure the correct column is used for each step of the procedure.</p>
<p> </p>
<p>All centrifugation steps are carried out in a benchtop microcentrifuge at 14,000 x g except where noted. Perform all centrifugation steps at room temperature.</p>
<p> </p>
<p>Ensure that all solutions are at room temperature prior to use.</p>
<p>It is important to work quickly when purifying RNA (see hints and tips on working with RNA at <a href="http://www.bioline.com/isolate" target="_blank">www.bioline.com/isolate</a>).</p>
<p> </p>
<p><strong>HANDLING AND STORING STARTING MATERIALS</strong></p>
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<p>RNA is not protected against digestion until the sample is flash frozen or disrupted in the presence of RNase inhibiting or denaturing reagents. Samples should be flash frozen in liquid nitrogen immediately and can be stored at -80°C for several months, or processed as soon as possible. Following disruption and homogenization in Lysis Buffer RX, samples can be kept at -80°C for up to one year, at 4°C for up to 24 hours or at room temperature for several hours. Frozen samples are stable for up to 6 months. Frozen samples in Lysis Buffer RX should be thawed slowly before starting the isolation of total RNA.</p>
<p> </p>
<p><strong>DISRUPTING AND HOMOGENIZING STARTING MATERIALS</strong></p>
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<p>For all RNA purification procedures, efficient disruption and homogenization of starting material is essential. To release all RNA contained in a sample, the total disruption of cell walls, plasma membranes and organelles must occur. Incomplete disruption results in reduced RNA yields. Homogenization reduces lysate viscosity following disruption and also facilitates efficient binding of RNA to the column membrane. Incomplete homogenization results in inefficient binding of RNA to the membrane and therefore reduced RNA yields.</p>
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<p><strong>Cells grown in a monolayer</strong></p>
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<p>Remove the cell culture medium completely. Incomplete removal of the medium will inhibit lysis of the cells and compromise the efficiency of RNA isolation. Wash with PBS and add Lysis Buffer RX immediately to the cell culture plate.</p>
<p> </p>
<p><strong>Cells grown in suspension</strong></p>
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<p>Centrifuge an appropriate number of cells and remove all supernatant by aspiration. Wash with PBS and lyse by adding Lysis Buffer RX.</p>
<p> </p>
<p><strong>Lifting of adherent cells with trypsin</strong></p>
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<p>To trypsinize adherent growing cells, first aspirate the cell culture medium. Add an equal volume of PBS to wash the cells and aspirate excess liquid. Add 0.1-0.3% trypsin in PBS to the washed cells. Incubate until the cells are detached. Add fresh, sterile culture medium and transfer cells to an appropriate tube (not supplied). Pellet cells by centrifugation for 10 min at 200 x g. Remove supernatant and add Lysis Buffer RX to the cell pellet.</p>
<p> </p>
<p><strong>Disruption using a mortar and pestle</strong></p>
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<p>An RNase-free mortar and pestle can be used in combination with liquid nitrogen to disrupt and lyse frozen or fibrous tissue samples, which are often solid. Grind the frozen tissue into a fine powder and add liquid nitrogen as necessary. It is important to ensure the sample does not thaw during or after grinding. After grinding, transfer tissue powder into a liquid nitrogen-cooled tube and allow the liquid nitrogen to evaporate. Add Lysis Buffer RX with reducing agent to the powdered tissue and mix immediately. Homogenize the sample with a nuclease-free 20 gauge (0.9 mm) syringe needle.</p>
<p> </p>
<p><strong>Disruption and homogenization using a rotor-stator homogenizer</strong></p>
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<p>Rotor-stator type tissue homogenizers can homogenize, disrupt and emulsify mammalian tissue samples in the presence of lysis buffer in seconds to minutes. Homogenization time depends on sample size and toughness. The spinning rotor disrupts and homogenizes the sample simultaneously by turbulence and mechanical shearing. Foaming can be minimized by keeping the rotor tip submerged. Select a suitably sized homogenizer: 5-7 mm diameter rotors can be used for homogenization in microcentrifuge tubes.</p>
<p> </p>
<p><strong>BUFFER PREPARATION AND PARAMETERS</strong></p>
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<p>Ensure 96-100% ethanol is available. Prepare the following:</p>
<p> </p>
<p><strong>Preparing Wash Buffer W1 with ethanol</strong></p>
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<p>Add 90 mL of 96-100% ethanol to Wash Buffer W1 Concentrate to give a final volume of 128 mL.</p>
<p><em>Note: Mark the label of the bottle to indicate ethanol was added. Store Wash Buffer W1 at room temperature (18-25°C).</em></p>
<p> </p>
<p><strong>Preparing Lysis Buffer RX with ß-mercaptoethanol (optional)</strong></p>
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<p>Optional: The use of ß-mercaptoethanol (ß-ME) in lysis is highly recommended for most mammalian tissues, particularly those known to have a high RNase content (e.g. pancreatic tissue). It is also recommended for users who wish to isolate RNA for sensitive downstream applications. Add 10 μL of ß-ME (provided by the user) to each 1 mL of Buffer RX required. ß-ME is toxic and should be dispensed in a fume hood. Alternatively, Buffer RX can be used as provided.</p>
<p><em>Note: TCEP can also be used as an alternative reducing agent. Use TCEP at a final concentration of 10 mM within Lysis Buffer RX.</em></p>
<p> </p>
<p><strong>Preparing DNase I (RNase-free) (optional)</strong></p>
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<p>Optional on-column digestion: For each on-column reaction to be performed, prepare a mix of 15 μL of DNase I Solution and 100 μL of DNase I Reaction Buffer DRB. Mix gently by inverting a few times.</p>
<p> </p>
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<p>Optional in-solution digestion: In a microcentrifuge tube, mix together 10 μL of DNase I Reaction Buffer DRB, 2.5 μL of DNase I Solution and up to 87.5 μL of RNA solution. For lower starting volumes of RNA, bring the volume up to 100 μL using RNase-free water. Gently swirl tube to mix solution.</p>
<p><em>Note: Do not vortex the DNase I as the enzyme is particularly sensitive to mechanical denaturation. Dispense into aliquots to avoid excessive freeze-thawing. Store aliquots at -20°C.</em></p>
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<p>Elute RNA using RNA Elution Buffer (included). The standard elution protocol can be modified for different applications.</p>
<ul>
<li>
<p>To achieve high yield, perform two successive elution steps with an elution volume described in the individual protocol (90-100% recovery rate). You may elute into the same or a different microcentrifuge tube depending on your application.</p>
</li>
<li>
<p>For both high-yield and high-concentration, elute with the standard elution volume. Then re-apply eluate onto the column for re-elution into the same microcentrifuge tube.</p>
</li>
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Always place eluted RNA on ice immediately to prevent degradation by RNases. For short-term storage freeze at -20°C but for long-term storage freeze at -80°C.</div>
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<div class="column"><strong>ELIMINATING GENOMIC DNA CONTAMINATION</strong></div>
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<div class="column">For most applications, genomic DNA is undetectable in preparations of RNA using the ISOLATE II Biofluids RNA Kit. Genomic DNA contamination is efficiently removed by the Genomic DNA Removal Column. However, if the sample contains high amounts of genomic DNA, additional treatment can be performed with the supplied DNase I. Two alternative protocols are provided. The on-column digestion protocol efficiently removes genomic DNA sufficient for most applications. For highly sensitive applications e.g. probe-based real-time PCR, a DNase I digest in the eluate can be performed to remove even traces of contaminating DNA. </div>
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<div class="column"><strong>TROUBLESHOOTING GUIDE</strong></div>
<div class="column">Please see the full Bioline manual.</div>
<div class="column"><a href="http://www.bioline.com/us/downloads/dl/file/id/3789/isolate_ii_biofluids_rna_kit_product_manual.pdf" target="_blank">http://www.bioline.com/us/downloads/dl/file/id/3789/isolate_ii_biofluids_rna_kit_product_manual.pdf</a></div>
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