biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsArno Veldstranscription profiling by arrayMus musculusMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-NCMF-33Gene expression of tumors derived from sorted Brca1/p53-deficient cell subpopulationsbiostudies-arrayexpressTumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivoLabeling - Hybridization of Oligo Microarrays with ULS-CY Dye labeled amplified RNA, using the TECAN HS4800 Hybridization Station. This protocol can be used to label amplified antisense RNA with the ULS system from KREATECH Biotechnology using the Amersham Cy Dyes (kit # EA-006). Make sure that the amplified RNA is as pure as possible! If Qiagen RNeasy columns are used in final purification step in amplification, make sure that no salts are left on the membrane of the column. This can be prevented by washing the RNA bound to the column twice with 80% ethanol, before eluting the aRNA. 1) For every reaction, pipet 2 ul 10X labeling solution into a 200 ul PCR tube. 2) Add 1 ug aRNA in a maximal volume of 17 ul. 3) Add 0.3 ul of ULS-Cy5 or 1 ul of ULS-Cy3 label. 4) Adjust the total volume to 20 ul with water and mix the solution by pipetting. 5) Incubate at 85 degrees C for 30 minutes. 6) Let the mixture cool to room temperature for about three minutes. 7) Shake a KREApure column (use a separate column for each reaction) to make sure that the column material is completely mixed (shake the material down before unscrewing the lid). 8) Break of the bottom part, remove the lid and place the column in a 2 ml Eppendorf vial without lid. 9) Spin the column for 1 minute at maximum speed. 10) Load the labeling mixture onto the column and place it in a clean 2 ml Eppendorf vial without lid, but keep the lid to close the vial after purification (make sure that you load the mixture on the highest part of the material: The fixed-angle rotor of the centrifuge forces the column material to one side of the column). 11) Place the highest part of the column material on the outside of the rotor and spin for 1 minute at maximum speed. 12) Discard the column and keep the eluate (about 20 ul). Analysis of labeled aRNA 13) Measure the amount of purified and labeled aRNA, and frequency of Dye incorporation (FOI) on the NanoDrop spectrophotometer with 2 ul of each labeled aRNA. FOI (number of Dye molecules per kb of aRNA) = (324.5 * pmol dye/ul) / RNA ng/ul 14) Pool the two purified reactions (total volume approximately 40 ul) and SpeedVac the mixture until a volume of 9 ul is reached. 15) Transfer the mixture to a 200 ul PCR tube and add 1 ul Fragmentation buffer to decrease the fragment size to 60-200 bases. 16) Incubate at 70 degrees C for 15 minutes. 17) Spin the vial briefly and add 1 ul of stop solution, mix by pipetting (the labeled aRNA can form aggregates which dissolve by pipetting) and place on ice until further use. 18) Transfer the mixture to a 1.5 ml Eppendorf vial and add 6 ul of blocking solution (containing 20 ug Poly d(A), 8 ug yeast t-RNA and 20 ug COT-1 DNA). 19) Add 43 ul of RNAse-free water and store at 42 degrees C. Note: If the labeled material is not used the same day for hybridization, it can be stored at -20 degrees C or -80 degrees C until further use. Samples can be stored for several weeks before use. If you are planning to do a large series of hybridizations in a short period of time, we recommend to first label all your aRNA before starting with the hybridizations. Used materials: ULS-Cy5* EA-006 KREATECH Biotechnology ULS-Cy3* EA-006 KREATECH Biotechnology KREApure column* EA-006 KREATECH Biotechnology 10x labeling Solution* EA-006 KREATECH Biotechnology *part of the kit RNA Fragmentation Reagents 8740 Ambion COT-1 DNA 15279-011 Invitrogen Poly d(A) 27-7836-01 Pharmacia Yeast t-RNA 109 495 RocheNucleic Acid Extraction - RNA Isolation with TRIZOL?? Reagent (Cell pellets(A), tissues(A) or cell culture dishes(B) (A)RNA isolation from cell pellets or tissues 1)Keep cell pellet or tissues at -80 degrees C before extraction. 2)Add TRIzol reagent while keeping the vial at -80 degrees C (Add 300 ul TRIzol reagent for 1*10E6 cells (a monolayer of cells on a large petridish is about 5*10E6 cells), for RNA isolation from tissues, use about 10 ml for each gram of tissue. 3)Homogenize the pellet by passing the lysate a few times through a pipette (do not vortex!). Tissue can be homogenized by using a Polytron. Make sure that all material has been homogenized - if not, this could result in degradation of the isolated RNA. Incubate for 10 minutes at room temperature before continuing the protocol. Continue with step 4 (B)RNA isolation from (10cm) cell culture dishes 1)Remove medium from dish and transfer 3-5 ml ice-cold PBS onto the dish. 2)Remove PBS and pipet another 3-5 ml PBS onto the dish, remove it and pipet 2-3 ml TRIzol reagent onto the dish. 3)Homogenize the cells by passing the lysate a few times through a pipette and transfer to a RNAse free tube. Make sure that all material has been homogenized - if not, this could result in degradation of the isolated RNA. Incubate for 10 minutes at room temperature before continuing the protocol. Continue with step 4 Continued extraction 4)Add 200 ul Chloroform for each ml of TRIzol reagent used for lysating cells or tissues and close the lid tightly and immediately shake vigorously up and down with force (do not vortex!) for 20 seconds and incubate for 10 minutes at room temperature. 5)Place vial in a refrigerated centrifuge (4 degrees C) and spin for 15 minutes at maximum speed (larger tubes can be spun for 45 minutes at 4000 rpm in a refrigerated (4 degrees C) swing bucket rotor). 6)Transfer he upper aqueous phase to a new vial, leave about 15-20% of the aqueous phase above the interphase to avoid contamination with proteins. 7)Add 500 ul of isopropyl alcohol for every ml of TRIzol reagent used for the initial homogenization, shake for 10 seconds and let precipitate for at least 15 minutes at room temperature (solution can be stored at -20 degrees C overnight). 8)Place vial in a refrigerated centrifuge (4 degrees C) and spin for 30 minutes at maximum speed (larger tubes can be spun for 45 minutes at 4000 rpm in a refrigerated (4 degrees C) swing bucket rotor). 9)A white pellet of precipitated RNA should be visible at the bottom of the tube (depending on the amount of cells/tissue as starting material). Note: A very pure RNA pellet can be transparent and therefore hardly visible. 10)Remove the supernatant (leave about 20 ul of the supernatant on the pellet, to avoid disturbance of the pellet) and add 1.5 ml 80% ethanol (with RNAse free water) to wash the pellet, agitate until pellet detaches from the bottom. 11)Place vial in a refrigerated centrifuge (4 degrees C) and spin for 10 minutes at maximum speed (larger tubes can be spun for 15 minutes at 4000 rpm in a refrigerated (4 degrees C) swing bucket rotor). 12)Remove the ethanol for as much as possible and dry the pellet by air (dry for about 10 minutes - make sure that the pellet does not dry completely, because this can result in a decreased solubility). 13)Dissolve the pellet in RNAse free water (use 50 ul RNAse free water for every 5*10E6 cells or 10 cm dish) measure RNA concentration at A260 (the A260/A280 ratio should be higher than 1.9). Used materials: TRIzol?? Reagent15596-026Invitrogen Life Technologies DNAse treatment of total RNA 1)Transfer the total RNA to a clean 1.5 ml tube. 2)Adjust the volume of the total RNA sample to 100 ul with RNAse free water. 3)Add 350 ul RLT buffer and mix by pipetting. 4)Add 250 ul 100% ethanol and mix by pipetting 5)Apply the sample to the RNeasy column and centrifuge for 30 seconds at 10.000 rpm (12.000 x g). 6)Discard flow-through and apply 350 ul RW1 buffer to the column and centrifuge for 30 seconds at 10.000 rpm (12.000 x g). 7)Discard flow through and place column back in the collection tube. 8)Transfer 70 ul of RDD buffer from the DNAse kit to a clean 200 ul PCR tube and add 10 ul DNAse from the same kit, mix this DNAse solution by pipetting (do not vortex). 9)Pipet the DNAse solution directly onto the center of the membrane (do not touch the membrane with the pipet tip). 10)Incubate for 15 minutes at room temperature. 11)Apply 350 ul RW1 buffer to the column and centrifuge for 30 seconds at 10.000 rpm (12.000 x g). 12)Discard flow through and collection tube and place column back in a clean collection tube. 13)Apply 500 ul 80% ethanol (with RNAse free water) and centrifuge for 30 seconds at 10.000 rpm (12.000 x g). 14)Discard flow-through and apply another 500 ul 80% ethanol (with RNAse free water) to the column and centrifuge for 30 seconds at 10.000 rpm (12.000 x g). 15)Discard flow through and place column back in the collection tube and centrifuge for 1 minute at maximum speed to completely dry the membrane of the column. 16)Transfer the column into a new 1.5 ml collection tube (comes with the RNeasy kit) and pipet 50 ul RNAse-free water directly onto the center of the membrane (do not touch the membrane with the pipet tip). Centrifuge for 1 minute at 10.000 rpm to elute. 17)Measure the amount and purity (the A260/A280 ratio should approximately between 2.1 and 2.3) of the cleaned RNA on the NanoDrop spectrophotometer with 2 ul 18)Run 1% agarose gel to check the RNA for degradation (use about 1 ug), the ribosomal bands should be well-defined, without visible smears. Used materials: Rneasy Mini Kit(250)74104Qiagen RNase-Free DNase Set (50)79254Qiagen T7-mRNA Amplification using the Invitrogen Superscript RNA Amplification System (#L1016-01) Note: Before starting with the protocol, the buffers used for purification have to be prepared. To the cDNA loading buffer, 3 ml of 100% isopropanol has to be added - to the cDNA wash buffer, 12 ml of 100% ethanol has to be added - to the aRNA wash buffer, 21 ml 100% ethanol has to be added. cDNA synthesis 1)Use 2-4 ug total RNA (preferably DNAse treated, especially if the A260/A280 ratio of the isolated total RNA is below 1.9. (Lower amounts of input RNA can be used - check the Invitrogen manual). 2)Make the following priming mix (total volume of 10 ul) in a 200 ul PCR tube: Total RNA (2-4 ug) max 9 ul T7-Oligo(dT) Primer 1 ul DEPC-treated water to 10 ul 3)Heat at 70 degrees C for 10 minutes, then put on ice. 4)Prepare the following first strand synthesis mix (total volume of 10 ul): 5x First Strand Buffer 4 ul 0.1 M DTT 2 ul 10 mM dNTP mix 1 ul RNaseOUT (40 U/ul) 1 ul Superscript III (200 U/ul) 2 ul 5)Add 10 ul of the first strand synthesis mix to 10 ul of the priming mix, mix by gently pipetting. 6)Incubate at 46 degrees C for 2 hours. 7)Incubate at 70 degrees C for 10 minutes. 8)Centrifuge the tube briefly to collect the contents and place the tube on ice. 9)Keep samples on ice until the second strand mix can be added. 10)Prepare the second strand synthesis mix (total volume of 130 ul): DEPC-treated water 91 ul 5x Second-Strand Buffer 30 ul 10 mM dNTP mix 3 ul DNA polymerase I (10 U/ul) 4 ul DNA ligase (10 U/ul) 1 ul RNAse H (2 U/ul) 1 ul 11)Add 130 ul of the second strand synthesis mix to the 20 ul of first strand synthesis solution and gently mix by pipetting. 12)Incubate at 16 degrees C for 2 hours. (The cDNA can now be stored at -20 degrees C). cDNA clean-up - Make sure that isopropanol is added to the cDNA loading buffer. - Transfer the cDNA mixture to a clean 1.5 ml tube. - Add 500 ul of cDNA loading buffer to the 150 ul of the cDNA mixture and mix by pipetting - Each Spin cartridge is pre-inserted into a collection tube. Load the cDNA mixture directly onto the spin cartridge. - Centrifuge at 10.000 rpm (12.000 x g) for 1 minute. - Discard the flow-through and place the Spin cartridge back into the collection tube. - Apply 700 ul of the cDNA wash buffer to the Spin cartridge and centrifuge for 2 minutes at 10.000 rpm (12.000 x g). - Discard the flow-through and place the Spin cartridge back into the collection tube. - Centrifuge for an additional 4 minutes at 10.000 rpm (12.000 x g). - Discard the flow-through and the collection tube. Place the Spin cartridge into a new recovery tube. - Apply 100 ul of DEPC-treated water to the center of the Spin cartridge and incubate for 2 minutes at room temperature. - Centrifuge at 10.000 rpm (12.000 x g) for 1 minute. Optional: Run 1% agarose gel to check cDNA product (use about 1/10 of the volume). A smear of cDNA should be visible with an average size of 1 kb. aRNA synthesis 13)SpeedVac the purified cDNA mixture until the volume is 23 ul. 14)Make an amplification mix, use the kit (total volume of 40 ul): cDNA 23 ul 100 mM ATP 1.5 ul 100 mM CTP 1.5 ul 100 mM GTP 1.5 ul 100 mM UTP 1.5 ul 10x T7 reaction buffer 4 ul (vortex well) T7 Enzyme mix 7 ul 15)Mix by gently pipetting, followed by briefly centrifuging to collect the contents of the tube. 16)Incubate at 37 degrees C for at least 6 hours (up to 9 hours - store at 4 degrees C as final step). 17)Store the amplification mix on ice until further use. aRNA clean-up - Make sure that ethanol is added to the aRNA wash buffer buffer before use. - Add 160 ul aRNA binding buffer to the aRNA mixture and mix thoroughly by pipetting (do not vortex). - Add 100 ul of 100% ethanol to the aRNA mixture and mix thoroughly by pipetting (do not vortex). - Each Spin cartridge is pre-inserted into a collection tube. Load the aRNA mixture directly onto the spin cartridge. - Centrifuge at 10.000 rpm (12.000 x g) for 15 seconds. - Discard the flow-through and place the Spin cartridge back into the collection tube. - Apply 500 ul of the aRNA wash buffer to the Spin cartridge and centrifuge for 15 seconds at 10.000 rpm (12.000 x g). - Discard the flow-through and place the Spin cartridge back into the collection tube. - Apply 500 ul of 80% ethanol (prepared with DEPC-treated water) and centrifuge for 15 seconds at 10.000 rpm (12.000 x g). - Discard the flow-through and place the Spin cartridge back into the collection tube. - Apply another 500 ul of 80% ethanol (prepared with DEPC-treated water) and centrifuge for 15 seconds at 10.000 rpm (12.000 x g). - Discard the flow-through and place the Spin cartridge back into the collection tube. - Centrifuge for 2 minutes at maximum speed to dry the column. - Discard the flow-through and the collection tube. Place the Spin cartridge into a new recovery tube. - Apply 100 ul of DEPC-treated water to the center of the Spin cartridge and incubate for 1 minute at room temperature. - Centrifuge at 10.000 rpm (12.000 x g) for 2 minutes. Analysis of aRNA 18)Measure the amount (the total aRNA yield should be between 40 and 80 ug) and purity (the A260/A280 ratio should approximately between 2.1 and 2.3) of the synthesized aRNA on a (NanoDrop) spectrophotometer (with 2 ul). 19)Run 1% agarose gel to check the labeled aRNA product (use about 1 ug). A smear of aRNA should be visible with an average size of 1 kb. Used materials: The SuperScript RNA Amplification System (#L1016-01) InvitrogenMIAME ScoreRaw DataOrganizationAssays and DataMAGE-TAB FilesArray DesignsArno VeldsSven RottenbergMarina Pajic, Ariena Kersbergen, Frank van Diepen, Anita Pfauth, Jos Jonkers, Piet Borst, Sven RottenbergfalseTranscription profiling by array of mouse tumours derived from sorted Brca1/p53-deficient cell subpopulationsGene expression of tumors derived from sorted Brca1/p53-deficient cell subpopulations2010-12-15T00:00:00Z2022-01-28T18:22:43.013Z2022-01-28T18:22:43.013ZE-NCMF-33EFO_0002768