STANDARD OPERATING PROCEDURE NHP GENOMICS CORE LABORATORY. QIAgen RNeasy Micro Protocol for Sorted cells

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1 STANDARD OPERATING PROCEDURE NHP GENOMICS CORE LABORATORY NHP Genomics Core Lab Subject: QIAgen RNeasy Micro Protocol for Sorted cells SOP Number: 44.0 Version: 1 Effective Date: December 8, 2007 Revised Date: October 6, 2008 QIAgen RNeasy Micro Protocol for Sorted cells Note: This protocol is adapted from the QIAGEN RNeasy Micro Handbook, Rationale: Small numbers of sorted cells, cultured cells and tissues may be disrupted in Qiagen RLT buffer purified using Qiagen RNeasy Micro kit. Materials: Materials Company Cat. No. QIAGEN RNeasy Micro Kit Buffer RLT QIAGEN Mercaptoethanol (>98%) molecular biology grade % Molecular Grade Ethanol SIGMA Various M ML QIAshredder columns RNAse free dh20 QIAGEN Various 1

2 Before Starting 1 RPE + ethanol 1 RPE + ethanol 2 80% EtOh Prepare 80% Ethanol by mixing 24 ml ethanol with 6 ml RNAse-free water (supplied). 3-70% EtOh Prepare 70% ethanol using % molecular volumes + RNAse free H20. Prepare enough to equal the volume of all samples (ie. 10 samples x 350 ul RLT = 3.5 ml 70% 4 - DNAse +RDD Prepare DNAse I RDD cocktail: per sample = 10 ul DNAse I + 70 ul RDD. Important points: To minimize expression changes induced by the sampling method, place the cells/tissue into the RLT buffer expeditiously. Observe general priniciples for handling RNA to maximize RNA integrity during sampling. See the links below for more information: Use RNAse-free, sterile polypropylene tubes for storing the sample. The amount of tissue to be used should be pre-determined on unimportant samples. PBMCs require 5-10x10 6 cells for consistently high yields of RNA. 1 cm gut biopsies yield approximately 5-10 ug of RNA. Some tissues require rigorous homogenization during sampling; the homogenization method should be predetermined on unimportant samples. 2-Mercaptoethanol (bme) should be added to RLT buffer at 1% volume before sampling (eg. 10 ml of bme per 1 ml of RLT). If bme cannot be used, use RLT alone, although yields may be lower. Use 350 or 600 ml of RLT per sample, unless the sample is unusually large/bulky and requires additional volume. For flow-sorted samples less than 500,000 cells, use 350 ul of RLT. Samples will be stored at -80 C, ensure that appropriate temperature resistant labels are used for sample tubes. RLT buffer is not suitable for stabilizing whole blood. Specific to the RNEasy Micro kit: 2

3 1. Recommended maximum for Micro kit is 5x10e5 cells, or 45 ug RNA. 2. Samples with < 500 cells are recommended to use carrier RNA (provided) HOWEVER this is incompatible with standard array amplification (Eberwine, Affymetrix) protocols that employ oligo-dt in the 1 st strand RT. Bacterial ribosomal RNA is recommended in this case. We do NOT use Bacterial ribosomal RNA because we cannot get accurate quantitation and quality read-out. 3

4 Protocol: 1. Harvest PBMCs. (maximum = 5x10e5 cells) Centrifuge PBMCs to pellet at 1500 rpm for 5 min. Aspirate supernatant. Note: For small samples (<500,000 cells) it can be difficult to see the pellets. In this case, we centrifuge at 600 x g for 10 min, and do NOT completely aspirate the supernatant, instead using excess RLT (350 ul) addition. 2. Add 75/350/650 ul of RLT buffer w. BME to sample, use additional volume if necessary. (for samples < 1x10e5 apply 75 ul RLT). Note: for small numbers of cells, we use 350 ul of RLT, and do not completely aspirate the sorting media (leave ul). 3. A - Homogenize the tissue as needed. For tissue culture cells and PBMCs, this requires only pipetting the samples several times, and vortexing at max. speed for s. Store the samples at -80 C. For small numbers of cells, do NOT use Shredder columns, they lose sample. 4. Add 1 volume 70% EtOh to lysate. Ie. 350 ul RLT = 350 ul 70% EtOh. 5. Transfer entire sample to RNeasy MinElute column within 2 ml collection tube. Spin for 15s - 1min at 8,000-10,000 rpm. Discard flowthru. 6. Wash with 350 ul RW1. Spin 15 s - 1 min at 8,000-10,000 rpm. 7. Make DNAse I + RDD cocktail (if not done already). Change collection column. 8. Apply 80 ul of DNAse I + RDD buffer cocktail to column. Incubate at RT for 15 min. 9. Wash with 350 ul RW1. Spin 15 s - 1 min at 8,000-10,000 rpm. 10. Wash with 500 ul RPE. Spin 15 s - 1 min at 8,000-10,000 rpm. 11. Wash with 500 ul 80% EtOh. Spin 15 s - 1 min at 8,000-10,000 rpm. 4

5 12. Spin MinElute column at 10,000 rpm with lid open for 5 min. While QIAGEN recommends full speed, columns have occasionally cracked at >12,000 rpm. 13. Place the RNeasy MinElute in a new 1.5 ml collection tube (labeled suitable for longterm storage). Apply 14 ul RNAse-free dh20 to the center of the membrane and spin for 2 min at 10,000 rpm to elute the sample. 14. Store at -80 C. 15. Measure yield/quality using NanoDrop & Agilent PICO RNA 6000 Kit. Note: for small numbers of cells (<100,000) Nanodrop cannot detect the low amount of RNA and is not recommended in favour of conserving sample. 5