5 min Cell/Virus RNA Extraction Kit

Transcription

1 5 min Cell/Virus RNA Extraction Kit Catalog #; R-1001 The fastest and the simplest but most reliable Nucleic Acid extraction system Kits designed for cellular RNA extraction from animal cell, bacteria, fungi, yeast, protozoa, and blood. Kit also designed for viral RNA extraction from serum, plasma, CSF, wash, culture media, nasal swab, and throat swab. Total procedure in 5 minutes No damage to RNA quality during extraction procedure Exclusive purification of RNA without any genomic DNA contamination Every procedure at ambient temperature without any cold or freezing step Single column format Final 5-20ug of cellular RNA or viral RNA for more than 20 reactions of RT-PCR or qrt-pcr reaction The kit can be stored at ambient temperature for more than a year. One kit for 50 RNA purification The extracted RNA will show around ratio of 260/280. The extracted RNA can be used in any downstream steps without any inhibitory effect. No Liquid nitrogen or an expensive homogenizing device 5 min Cell/Virus RNA Extraction Kit

2 The visual manual is available on Youtube: Storage Conditions and Product Stability All components should be kept tightly sealed and stored at room temperature. These reagents should remain stable for at least 1 year in their unopened containers. Precautions and Disclaimers This kit is designed for research purposes only. It is not intended for human or diagnostic use. Customer-Supplied Reagents and Equipment Benchtop microcentrifuge 1.5 ml microcentrifuge tubes Vortex % isopropanol % ethanol Sterile cotton swab and scissors (for nasal and throat swab preparation) Kit components for 1 box/50 prep Component Column/collection tube Cell/Virus RNA Solution RNA Washing Solution RNA Elution Buffer Product insert Products 50 ea. 25 ml 18 ml 7.5 ml 1 ea. 5 min Cell/Virus RNA Extraction Kit

3 5 min Cell/Virus RNA Extraction Kit Before Starting 1. Add 42 ml of % ethanol to RNA Washing Solution. Mix well. Mark on the labels that ethanol is added. Store it at room temperature. 2. Add 35 ml of % isopropanol to Cell/Virus RNA Solution. Mix well. Mark on the labels that isopropanol is added. Store it at room temperature. Procedures for lysate preparation for Cell/Virus RNA extraction Notice; All centrifugation is in 10,000 rpm at room temperature. All steps at ambient temperature A. Cultured animal cells General notice for lysate preparation 1. The maximum recommended input of cells is 2.5 x As a reference, confluent 60mm and 100mm dish contain 3.2 X10 6 and 8.8 X10 6 cells respectively. 2. The lysate can be stored at -20 o C for later RNA extraction. 3. For RNA extraction from frozen cell, loosened the pellet by scraping the tube for 5-6 times over an uneven surface such as a microcentrifuge tube rack and add lysis solution directly to frozen sample. Lysate preparation I. Cell grown in monolayer 1. Aspirate media 2. Add 700 ul of Cell/Virus RNA Solution and mix with gentle swirling for 30 sec. 3. Transfer all lysate to a microtube and vortex for 15 sec. 5 min Cell/Virus RNA Extraction Kit 3

4 II. Cell grown in suspension/ detached monolayer cells /milk 1. Transfer cell suspension to a microtube (not provided) and centrifuge for 30 sec. 2. Remove supernatant with pipetting or a gentle vacuum. 3. Repeat steps 1 and 2 as required. 4. Closed the cap and loosened the pellet by scraping the tube for 5-6 times over an uneven surface such as a microcentrifuge tube rack. 5. Add 700 ul of Cell/Virus RNA Solution and vortex for 30sec. B. Blood sample General notice for lysate preparation 1. Blood sample should be fresh and used within a couple of hours after collection. 2. Frozen blood sample is not appropriated for blood RNA extraction by this kit. Lysate preparation 1. Invert mix for 5-6 times of whole blood in EDTA collection bottle. 2. Transfer 200 ul of whole blood into a microtube. 3. Microcentrifuge for 30 sec. and remove all liquid. 4. Closed the cap and loosened the pellet by scraping the tube for 5-6 times over an uneven surface such as a microcentrifuge tube rack. 5. Add 700 ul of Cell/Virus RNA Solution and vortex for 30sec. C. Bacteria, fungi, yeast, and protozoa General notice for lysate preparation 1. It is recommended that no more than 1.5 ml of saturated bacterial or yeast culture volume be used in this procedure in order to prevent clogging of the column. 2. It is recommended that no more than 100 mg of fungi or protozoa be used for this procedure in order to prevent clogging of the column. Lysate preparation 1. Transfer cell in culture media or PBS to a microtube (not provided) and centrifuge for 30 sec. 2. Remove supernatant with pipetting or a gentle vacuum. 5 min Cell/Virus RNA Extraction Kit 4

5 3. Closed the cap and loosened the pellet by scraping the tube for 5-6 times over an uneven surface such as a microcentrifuge tube rack. 4. Add 700 ul of Cell/Virus RNA Solution and vortex for 30sec. D. Viral RNA from serum, plasma, CSF, wash, and media Lysate preparation for viral RNA 1. Transfer less than 350 ul of sample to a microtube 2. Add 1 ml of Cell/Virus RNA Solution and vortex mix for 30 sec. E. Nasal or Throat Swabs Lysate preparation 1. Transfer 700ul of Cell/Virus RNA Solution to a clean microtube (not provided) 2. Gently brush a sterile, single-use cotton swab (not provided) inside the nose or mouth of the subject. 3. Cut the cotton tip of swab with clean scissors and leave in the microtube. 4. Closed the cap and vortex for 30 sec. 5 min Cell/Virus RNA Extraction Kit 5

6 Procedures for Cell/Virus RNA Extraction from the lysate Important notice; All centrifugation in 10,000 rpm at room temperature. If column is clogged, spin more until a complete flowthrough. Universal procedure for all lysate All steps at ambient temperature 1. Transfer all lysate to column. Do not centrifuge to collect the sample from the lid. Any centrifuge in this step would severely reduce the amount of purified RNA. 2. Centrifuge the column for 15 sec. 3. Discard the flowthrough. Reassemble the spin column with its collection tube. 4. Repeat the steps of 1 and 3 if required. 5. Apply 700 ul of RNA Washing Solution and centrifuge for 15 sec. Discard the flowthrough. 6. Apply 400ul of RNA Washing Solution and centrifuge for 30 sec. Discard the flowthrough. 7. Replace the collection tube with a clean microcentrifuge tube (not supplied). 8. Add 100ul of RNA Elution Buffer to column. 9. Close the cap and vortex for 15 sec. 10. Centrifuge for 15 sec. 11. Reload the flowthrough to the top of column and centrifuge for 30 sec. 5 min Cell/Virus RNA Extraction Kit 6

7 Example of test The RNA extracted by 5 min Cell/Virus RNA Extraction Kit is free from genomic DNA contamination. Total RNA was purified from 6 different HEK 293 cultured cells using the kit. RNA was extracted from 1 X 10 6 cells and eluted to 100 ul of Elution Buffer. Ten ul each RNA was analyzed on 1 % agarose gel. Lane 1 to 6; Before RNAse treatment, Lane 7 to 12; after RNase treatment. Comparison of cellular RNA extracted by two different RNA extraction Kits. RNA was extracted from 1 X 10 6 HeLa cell either by RNeasy mini Kit (Qiagen, lanes 1 and 2) or 5 min Cell/Virus RNA Extraction Kit (Biofactories, lanes 3 and 4). Ten ul of final Elution buffer was analyzed on 1 % agarose gel. 5 min Cell/Virus RNA Extraction Kit 7

8 An exclusive extraction of RNA by 5 min Cell/Virus RNA Extraction Kit. Total 2 X 10 6 MDB- MB-468 and HeLa cell was used for genomic DNA extrafction by 5 min Cell/Virus DNA Extraction (lane 1 and 3) and total RNA extraction by 5 min Cell/Virus RNA Extraction Kit (lane 2 and 4). Five ul of each Nucleic acid was analyzed on 1 % agroase gel. Reproducibility of Cell/Virus RNA Extraction Kit. Total 6 independent samples from MDB-MB- 468 cell (2 X 10 6 ) or HeLa cell (2 X 10 6 ) were processed with 5 min Cell/Virus RNA Extraction Kit. Five ul of each eluted RNA was loaded on 1 % agarose gel. Lanes 1-6; from MDB-MB-468 cell, lanes 7-12; HeLa cells. Reproducibility of Cell/Virus RNA Extraction Kit. Total13 independent RNA preparation was 5 min Cell/Virus RNA Extraction Kit 8

9 performed using an identical E. coli culture by 5 min Cell/Virus RNA Extraction Kit. Total 1.5 ml of saturated E. coli culture was used for each preparation. Ten ul of each eluted RNA was loaded on 1 % agarose gel. Blood RNA extraction by 5 min Cell/Virus RNA Extraction Kit. Six blood samples were used for RNA extraction by Cell/Virus RNA Extraction Kit within an hour of collection. Ten ul of final Elution Buffer was analyzed on 1 % agarose gel. Bacterial RNA extraction by 5 min Cell/Virus RNA Extraction Kit. Ten ul of final Elution Buffer was analyzed on 1 % agarose gel. Lane 1; from 0.5 ml, lane 2; 1 ml, and lane 3; 1.5 ml of saturated E. coli culture 5 min Cell/Virus RNA Extraction Kit 9

10 Comparison of different viral RNA extracted by two different RNA extraction Kits. EMCV virus was infected to HeLa cells on 100mm plate with about 70% confluence. After 4 days, all supernatant was harvest and used for viral RNA extraction as described in each protocol from RNAeasy mini Kit (Qiagen) or 5 min Cell/Virus RNA Extraction kit. The amount of the viral RNA was determined by qrt-pcr using EMCV specific primers in CFX96 (Biorad). In each assay 5 ul of RNA was used by qrt-pcr pre-mixture from Takara. Each value represents the average of two independent assays. Samples RNeasy Mini Kit 5 min Cell/Virus RNA Kit Two hundred fifty ul of milk was spiked with the indicated volume of statured E. coli culture and used for total RNA extraction using 5 min Cell/Virus RNA Extraction Kit. Lane 1; marker, lane 2; no spike, lane 3; 2.5 ul of E. coli culture, lane 4; 20 ul, lane 5; 50 ul, and lane 6; no milk and only 50 ul of the culture. 5 min Cell/Virus RNA Extraction Kit 10

11 Total RNA prepared from frozen HeLa cell by the 5 min Cell/Virus RNA Extraction Kit is completely free from any contamination of genomic DNA. Total RNA was extracted with three different RNA extraction products (Lanes 1 and 2; from company Q, lanes 3 and 4; company R, and lanes 5 and 6; from Biofactories). Ten ul of eluted sample was incubated in the presence of 2 ul of DNAse free RNAse (NEB) for 5 minutes and loaded on 1 % agarose gel. Technical Support Biofactories Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of our products. If you have any questions or experience any difficulties regarding products, please do not hesitate to contact us. For technical assistance and more information, please contact our Technical Support Team through at techsupport@5mindna.com. 5 min Cell/Virus RNA Extraction Kit 11