Manual - Trimgen
Contents
1. WaxFree RNA User Manual Frequently Asked Questions Q Can I use the kit to extract RNA from old paraffin sample A Yes We successfully extracted RNA from 10 year old paraffin sample and the RT PCR results were excellent Q Can I use the kit to extract DNA A Yes The final extract includes RNA and DNA from tissue Q Can I amplify 300bp product from the RNA sample A No The RNA in the paraffin sample is already partially degraded during formalin fixation The average size of RNA is about 100 120 bp Q Can I design the PCR primers in same exon A No If the PCR primers are located in same exon it could amplify the genomic DNA instead of RNA because most RNA extraction methods have DNA contamination To avoid the artificial DNA amplification you should design the forward primer in one exon and the reverse primer in next exon TrimGen Corporation WaxFree RNA User Manual Kit Contents The kit provides extraction reagents for 50 samples Component Quantity Q Solution 50 ml Wash Buffer 60 ml Enzyme Mix 0 5 ml R Resin 7 5 ml WR Filter 50 Materials and Equipment Needed 2ml sterile screw cap microcentrifuge tubes Laboratory incubator Heat Block 1 2 Vortex Mixer Microcentrifuge 70 Ethanol optional for Standard Protocol ONLY RNase Free DNase is not included in the kit The enzyme can be purchased separately from TrimGen Cat No DE 50 or from other vendors TrimGen Corporat2. final extract to the RT PCR reaction However the excess of final extract may inhibit the RT PCR reaction It is necessary to titrate the final extract for the RT PCR reaction Less RNA released For some tissues such as skin and muscle the 1 hour enzyme digestion is not long enough to extract RNA efficiently from these tissues Increasing the enzyme digestion time to 3 hours or overnight at 45 C will increase the yield of RNA Some of the R resin are leaking to the collection tube Can use the final extract to perform the RT PCR Yes You need to spin the collection tube at 10 000 x g to bring down the R resin and then use the supernatant Check your centrifuge to reduce the spin speed to 1 000 x g The RNA concentration calculated from the OD 60 280 readings is high and RT PCR does not work RNA concentration is too high Dilute the extraction supernatant with nuclease free water then perform the RT PCR PCR amplicon size is too big The formalin fixation will damage RNA The average size of RNA in the FFPE sample is about 100 150 bp When the designed amplicon size is too big the PCR may not work because the genome RNA in the FFPE tissue already broken The RNA quality depends on the tissue type storage time and fixation conditions Our customers have successfully amplified 150 bp PCR products from 10 year old samples 15 16
3. solution in the tube is the final extract which contains RNA ready for reverse transcription reaction or one step RT PCR Note Storage of RNA is not recommended even at 80 C The extracted RNA should be converted to cDNA after extraction The cDNA can be stored at 20 C 23 RNA Concentration Measurement see page 11 TrimGen Corporation WaxFree RNA User Manual Short Protocol Pre heat a heat block or incubator to 45 C Set another heat block to 90 C 1 Collect 1 5 or 2 ml tubes screw cap and label the tubes with the sample ID Collect sample Paraffin section on slide One section with tissue size 1 2 cm and 5 20um thicknesses Scrape the tissue from slide and transfer to a tube Paraffin tissue block Trim away surrounding paraffin Cut and transfer 10 30 mg of tissue to a tube Fine needle aspiration sample transfer entire sample to a tube Fresh or frozen tissues Cut and transfer 10 30mg tissues to a tube Culture cells Transfer 100 ul cell suspension 10 10 cells to a 2 ml tube For solid tissue with high cell density such as brain or liver reduce the amount of tissue for extraction Blank control If you are planning to measure the RNA concentration after the extraction you need to set up a Blank Control tube Add an extra tube to Step 2 and process it as a sample but without adding any tissue This tube is the Blank Control for ODz 9 280 Calibration 3 Re suspe
4. TrimGen Corporation WaxFree RNA User Manual Downstream Applications Reagents are not included in this kit Reverse transcription Use 10 25ul of the final extracts as template for total 50ul of reverse transcription reaction The reverse transcribed cDNA can be stored at 20 C PCR amplification The PCR enzyme and condition varies in different laboratories Before starting routine operation we recommend that the first time user perform a sample titration test to find a proper sample amount for your PCR amplification As an example use 5ul 10ul 15ul of RT products for 25ul PCR reactions to determine the best sample volume for PCR The forward and reverse PCR primers should be designed in different exons to prevent the artificial amplification of genomic DNA One Step RT PCR Use 5 10 ul of the final extraction supernatant as a template for total 50 ul of One step RT PCR reaction Sample normalization for quantitative PCR The OD 260 absorbance of final extraction can be used as a reference to normalize the difference between samples 13 TrimGen Corporation WaxFree RNA User Manual Troubleshooting Guide Problem Suggestions The removal of paraffin is incomplete Too much tissue sample WaxFree RNA standard protocol is optimized for a maximum of 3 FFPE sections 5 20 um thick up to a 3 cm each Trim off excess paraffin before extraction For larger sections reduce the number of se
5. TrimGen Corporation WaxFree RNA User Manual Limited Product Warranty It is imperative that the users strictly adhere to this manual Failure to CONTENTS do eo il void TrimGen s guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose Introduction 3 Notice to Purchaser i The product is provided as Research Use Only Not for use in Frequently Asked Questions 4 diagnostic procedures The purchaser must determine the suitability of the product for their particular use Kit Contents 5 The purchase of WaxFree RNA products includes a limited nonexclusive license to use the kit and systems This license does Materials and Equipment Needed 5 not grant rights to use the kit and systems for reproduction of the WaxFree RNA kit and systems to modify the WaxFree RNA kit and systems for resale or to use the WaxFree RNA kit and systems Standard Protocol 6 to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by Short Protocol 9 estoppels is granted Product Safety and Liabilities Downstream Applications 10 When working with the kit reagents always wear a suitable lab coat disposable gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental dam
6. ages arising out of the misuse the results of use or the inability to use this product Storage Upon receipt store the kit at 2 8 C Shelf Life Storage options Shelf life Kits stored at 2 8 C 6 months Enzyme Mix stored at 20 C All other components stored at 2 8 C Veet TrimGen Corporation WaxFree RNA User Manual Introduction WaxFree RNA kit is designed to extract RNA from 1 Formalin fixed paraffin embedded FFPE tissue 2 Fine needle aspiration FNA samples 3 Fresh or frozen tissue 4 Cells WaxFree RNA kit is a homogenous extraction method which eliminates RNA loss inherant in traditional column beads and phenol chloroform extraction methods The R resin and the enzyme mix are optimized to maximally release RNA from tissues to increase the yield of RNA The extracted RNA from one paraffin section size 1 x 1cm 10um thickness is sufficient to perform up to 15 RT PCR reactions The kit uses Q Solution a non toxic solution to efficiently remove paraffin and formalin residual from tissue The WaxFree RNA kit can be used with the Standard Protocol or Short Protocol The applications for each protocol are listed in the table below Tissue Type Standard Protocol Short Protocol FFPE Slides Vv V v Tissue dissected from x paraffin slide or block v Fine needle aspiration FNA sample x y Fresh or frozen tissues x vV Culture cells x V TrimGen Corporation
7. areful not to disturb the pellet 13 Re suspend R Resin thoroughly by shacking the bottle several times Transfer 120 ul 60 ul for small paraffin tissue or FAN sample of the R Resin to each tube The step 14 and 15 are designed for skin or muscle tissues only for other type of tissues go to step 16 14 Cap the tube and vortex 10 seconds at high speed 15 Incubate the tube at 90 C for 10 minutes then cool the tube to room temperature 20 25 C 16 Add 7 ul 3 5 ul for small paraffin tissue or FAN sample of Enzyme Mix to each tube Cap the tube and mix the content by flicking the tube For multiple samples Prepare pre mixed R Resin with Enzyme Mix using formula below R Resin Sample x 120 60 for small tissue x 1 1 Enzyme Mix Sample x 7 3 5 for small tissue x 1 1 Transfer 127ul 63ul for small size tissue of the mixture to each tube Return the Enzyme Mix to 20 C for storage 17 Incubate the tube at 45 C for 1 hour 18 Heat the tube at 90 C for 10 minutes TrimGen Corporation WaxFree RNA User Manual 19 Place WR Filter into a 1 5 ml tube and label the tube with sample ID Make sure the white filter is at the bottom of the column see below White filte White filter 20 After incubation transfer the entire extraction mix to WR filter 21 Centrifuge the tube at 1 000 x g about 1500 3 000 rpm in most tabletop centrifuge for 3 minutes 22 Discard the WR Filter The
8. ctions used for extraction Add more Q solution for de paraffinization The final RNA extract has yellow or brown color the RT PCR does not work well The paraffin samples such as bone marrow spleen and liver contains high blood component The hemoglobin in the sample is the cause of the color and also inhibits the RT or PCR reaction It is necessary to remove these molecules by further purification using a spin column TrimGen Spin 50 Cat No TC 50 or other commercial RNA purification kit The O D260 280 ratio is below our QC criteria can use the extracted RNA for RT PCR Yes The RNA quality assessment is different from conventional extraction methods A typical OD2z 0 280 ratio for the WaxFree ranges from 0 6 to 7 5 The low OD ratio will not affect the RT PCR reaction For special applications such as a microarray study further purification may necessary The OD 260 is too high Need Blank Control tube Use the Blank control tube to calibrate the spectrophotometer then measure 14 TrimGen Corporation WaxFree RNA User Manual TrimGen Corporation WaxFree RNA User Manual the OD For larger size tissue sections or solid tissues with high cell density the final RNA extract needs to be diluted with water The RNA concentration calculated from the OD 60 280 readings is low and the RT PCR does not amplify properly The RNA concentration is low Add more
9. ion WaxFree RNA User Manual Standard Protocol Pre heat a heat block or incubator to 45 C Set another heat block to 90 C 1 Collect 1 5 or 2 ml tubes screw cap and label the tubes with sample ID 2 Collect sample Paraffin section on slide One section with tissue size 1 2 cm and 5 20 um thickness Scrape the tissue from slide and transfer to a tube Paraffin section prepared in tube directly go to step 3 Paraffin tissue block Trim away surrounding paraffin Cut and transfer 10 30mg of tissue to a tube Fine needle aspiration sample transfer entire sample to a tube For solid tissue with high cell density such as brain or liver reduce the amount of tissue for extraction Add 0 8 ml of Q Solution to each tube Screw cap on and vortex for 30 seconds at high speed 5 Incubate the tube at 45 C for 20 minutes 6 Vortex the tube 30 seconds at high speed 7 Centrifuge the tube at 10 000 x g about 12 000 14 000 rpm in most tabletop centrifuge for 10 minutes 8 Discard the supernatant using a pipettor or by aspiration Be careful not to disturb the tissue pellet 9 Add 1 ml of Wash Buffer to the tube 10 Screw cap on and vortex 10 seconds at high speed to re suspend the pellet TrimGen Corporation WaxFree RNA User Manual 11 Centrifuge the tube at 10 000 x g about 12 000 14 000 rpm in most tabletop centrifuge for 10 minutes 12 Discard the supernatant using a pipettor or by aspiration Be c
10. nd the R Resin by thoroughly shaking the bottle several times Then transfer 120ul of the R Resin to each tube for small size tissue add 60ul see table 1 TrimGen Corporation WaxFree RNA User Manual 4 Add 7 ul of Enzyme Mix to each tube for small size tissue add 3 5nl see table 1 Table 1 Tissues Size PRESINI acu FFPE on slide 0 5 2 cm 120 7 FFPE on slide lt 0 5 cm 60 3 5 FNA 60 3 5 Fresh tissue 10 30 mg 120 7 Fresh tissue lt 10 mg 60 3 5 Cells gt 10 10 120 7 Cell lt 10 60 3 5 For multiple samples Prepare pre mixed R Resin with Enzyme Mix using formula below R Resin Sample x 120 60 for small tissue x 1 1 Enzyme Mix Sample x 7 3 5 for small tissue x 1 1 Transfer 127ul 63ul for small size tissue of the mixture to each tube 5 Screw cap on and mix the content by flicking the tube Incubate the tube at 45 C for 1 hour 7 Heat the tube at 90 C for 10 minutes Return the Enzyme Mix to 20 C 10 TrimGen Corporation WaxFree RNA User Manual 8 Place WR Filter into 1 5 ml tubes and label the tubes with sample ID Make sure the white filter is at the bottom of the column see below White filte White filter 9 After incubation transfer the extraction mix to WR filter 10 Place the WR Filter in a new tube and centrifuge the tube at 1 000 x g about 1 500 3 000 rpm in most tabletop centrifuge for 3 minutes 11 Discard the WR Filter The soluti
11. on in the tube is the final extract which contains RNA ready for reverse transcription reaction or one step RT PCR Note Storage of RNA is not recommended even at 80 C The extracted RNA should be converted to cDNA after extraction The cDNA can be stored at 20 C 11 TrimGen Corporation WaxFree RNA User Manual RNA Concentration Measurement Measure by OD2 method Aliquot 5ul of final extract to new tube Dilute the final extract with 45ul water Calibrate the UV spectrophotometer using the diluted Blank Control and adjust the OD2g and OD2 9 to zero to remove any background absorbance caused by the reagents Then measure the diluted samples at OD260280 to calculate the RNA concentration The following equation can be used to determine the concentration of the extracted RNA A convenient calculation form is available online www trimgen com wf DNA calculation xls RNA Conc ng l 62 9 x OD269 36 x OD2s0 x dilution factor 0 5 RNA Quality WaxFree is a homogeneous extraction method The proteins from tissue remain in the final extracts and cause a reduction in the OD ratio A typical OD260 280 ratio ranges from 0 8 to 1 3 The low OD ratio will not affect the reverse transcription or RT PCR Measure by fluorescent method The RNA concentration can be accurately measured by RiboGreen method Invitrogen Cat No R R 32700 http probes invitrogen com media pis mp32700 pdf 12
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