Affymetrix GeneChip Drosophila Genome [DrosGenome1]

Function-informed transcriptome analysis of Drosophila renal tubule

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The experiment was designed to identify genes abundant or enriched in adult Drosophila melanogaster Malpighian (renal) tubule as compared to the whole fly.

Transcription profiling of adult drosophila renal tubule vs whole fly

The experiment was designed to identify genes abundant or enriched in adult Drosophila melanogaster Malpighian (renal) tubule as compared to the whole fly.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

Affymetrix:Protocol:Percentile

Title: Affymetrix CEL Analysis (Percentile). Description:

P-MEXP-4217

Total RNA Extraction by TRI Regent  1. 7 Days whole and tubules in 1.5ml tubes. 2. Flash freeze tubes in liquid nitrogen then store in –80°C freezer until ready for RNA extraction. 3. Place sample on ice and add 1ml TRIzol (Sigma) per 100mg tissue (Or add just a third of this volume to make thawing and homogenisation easier, and make up to the total volume after homogenisation). 4. Homogenise the sample for 1 min at full speed.( eg using a Ultra-Turrax T8, IKA). 5. Increase volume of homogenised sample to the recommended level with TRIzol and incubate for 5 min at RT. 6. Centrifuge at 11,000g for 10 mins at 4°C to pellet much of the chotion, vitelline membranes, cuticle etc. Transfer the supernatant to a new 1.5ml RNAse free tube. 7. Add 0.2ml of chloroform per 1ml TRIzol, shake vigorously for 15sec and incubate at RT for 2-3 mins.  8. Centrifuge at 11,000g for 15mins at 4°C. 9.  Remove upper phase to a new tube being careful not to touch the interface. 10. Add 0.7 volumes of isopropanol to precipitate RNA. Incubate at RT for 10min and then centrifuge at 11,000g for 10min at 4°C. 11. Discard the supernatant and wash the RNA pellet with 1ml 70% ethanol/Nuclease free water per 1ml of the original TRIzol volume. Centrifuge at 11,000g for 10min at 4°C. 12. Air dry the pellet briefly. Resuspend in an appropriate volume of Nuclease free water. 13. Verify quality of RNA on an agarose gel and measure the OD260 to estimate concentration.   RNA clean up by  Qiagen Kit  1. Adjust sample to a volume of 100ul with RNAse-free water, add 350ul Buffer RLT to the sample and mix thoroughly.  Ensure beta-ME has been added to RLT. 2. Add 250ul ethanol (96-100%) to the lysate and mix well by pipetting. DO NOT CENTRIFUGE. 3. Apply sample (700ul) to a Rneasy minispin column sitting in a collection tube. 4. Centrifuge for 15 secs at full speed 5. Reapply the flow through to the column and repeat centrifugation. 6. Discard flow-through and collection tube. 7. Transfer the Rneasy column into a new 2ml collection tube. Add 500ml Buffer RPE and centrifuge for 15 seconds at full speed to wash. 8. Discard flow-through and re-use the collection tube in next step. 9. Pipet 500ul Buffer RPE onto Rneasy column and centrifuge for 2 minutes at full speed to dry the Rneasy membrane. 10. Discard the filtrate and centrifuge the Rneasy spin column at full speed for 1 minute.  11. Transfer the Rneasy column into a new 1.5ml collection tube and pipette appropriate volume of Rnase-free water onto membrane (30ul). Leave to stand for 1min and centrifuge for 1 min at full speed to elute (label E1), store E1 on ice.  12. Repeat elution with as second 30ul into a fresh 1.5ml tube (label E2), store on ice. 13. Run the elution E1 on Agilent Bioanalyzer to check the quality and the quantity. 14. If  the concentration is less than 500ng/ul. We will use pellet paint precipitation to get the right concentration.   PELLET PAINT  PRECIPITATION  1. Thaw the Pellet Paint and 3M Na Acetate (pH 5.2) and bring to room temperature.  2. Add 1ul Pellet Paint to the sample, followed by 0.1 volume of 3M Na Acetate. 3. Add 2 volumes of absolute ethanol and vortex briefly 4. Incubate at room temperature for 2 minutes. 5. Spin sample in a microcentrifuge at top speed for 5 minutes. 6. A coloured pellet should be visible at the bottom of the tube, remove as much of the supernatant as possible without disturbing the pellet. 7. Wash the pellet with 70% ethanol, vortex briefly and spin as in step 5. 8.  Wash the pellet with 100% ethanol, vortex briefly and spin as in step 5. 9. Remove supernatant and air dry pellet.  10. Resuspend pellet in an appropriate volume of Nuclease-free water. 

P-MEXP-4222

SYNTHESIS OF BIOTIN LABELLED cRNA  ENZO KIT (ONLY FOR CUSTOMERS THAT NEED TO COMPARE THEIR SAMPLES WITH PREVIOUSLY RUN SAMPLES USING THIS METHOD)  In table 6 the correct amounts of cDNA and H2O to use for the reaction are listed depending from the starting amount.  Table 6 Total RNA (ug) Volume of cDNA (ul) Volume of H2O(ul) 2.0 – 4.9 12 10 5.0 – 8.0 10 12 8.1-16.0 5.0 17  Table 7 Reagent Volume Template cDNA Variable see above (table 6, column 2) RNase-free H20 Variable see above (table 6, column 3) 10 x HYB Reaction Buffer 4ul 10 x Biotin-labelled Ribonucleotides 4ul 10 x DTT 4ul 10 x RNase Inhibitor mix 4ul 20 x T7 RNA Polymerase 2ul   Total Volume 40ul  Procedure  1. Add all reagents then spin the tube to collect all liquid @ the bottom 2. Place in any PCR machine, use program AFFY37, the program will run for 4-5 hrs @ 37 C  It is possible to store @ –20 C if the labelled cRNA is not purified immediately. 

Transcription profiling of adult drosophila renal tubule vs whole fly

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