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ARK Genomics

Standard Operating Procedure

S.O.P#

IGF100.00

Title

Isolation of total RNA.

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1.0	BACKGROUND Extraction of good quality total RNA is vital to the production of high quality expression data. We suggest that you take all the usual precautions during the preparation of the RNA to prevent degradation or contamination.
2.0	PURPOSE This protocol describes the method of total RNA isolation required by ARK Genomics. We have found that the double extraction method using first Trizol and then RNeasy significantly reduces the levels of background after hybridization on a microarray. Some samples may fail to label if only a single extraction has been carried out.
3.0	TRIZOL PROTOCOL Homogenization
	3.1	Add 1ml of Trizol to 100mg of tissue.
	3.2	Homogenise in a polytron at half speed for 2 minutes. (For tough tissue increase the speed and time: CARE)
	Phase Separation
	3.3	Incubate at room temperature for 5 minutes.
	3.4	Add 0.2 ml chloroform, shake tube vigorously by hand for 15 seconds.
	3.5	Incubate at 15 - 30ºC for 2 - 3 minutes.
	3.6	Centrifuge samples at 12000 x g for 15 minutes at 2 - 8ºC.
	3.7	Transfer aqueous (upper) phase to a new tube (take only 500ul). [The upper phase is 60% of the volume of Trizol used for homogenization.]
	RNA precipitation
	3.8	Add 500ul of isopropyl alcohol.
	3.9	Incubate for 10 minutes at 2 - 8ºC.
	3.10	Centrifuge at high speed for 10 minutes at 2 - 8ºC. The RNA precipitate forms a gel like pellet on the side/bottom of the tube.

	RNA Wash
	3.11	Remove the supernatant and wash the pellet with 1ml of 75% ethanol.
	3.12	Vortex the sample and then centrifuge for 5 minutes at 2 - 8ºC (>10000 x g)
	3.13	Remove the supernatant and air dry the RNA pellet for 5 - 10 minutes.
	Redissolving the RNA
	3.14	Resuspend in RNase free water. Adjust sample to 100ul volume.
4.0	RNeasy PROTOCOL
	Before proceeding with this protocol it is necessary to add b-mercaptoethanol to the RLT buffer. In a fume hood with appropriate protective clothing add 10ul of b-mercaptoethanol per 1ml of RLT buffer. This buffer is stable for one month after preparation. Ensure ethanol has been added to the RPE buffer before use. Add 4 volumes of 100% ethanol (as indicated on the bottle) before use.
	4.1	Add 350 ul RLT buffer (with b-mercaptoethanol) to the 100ul RNA sample, mix thoroughly.
	4.2	Add 250ul of 100% ethanol and mix well by pipetting 10 times.
	4.3	Apply the sample (700ul) to an RNeasy minicolumn placed in a 2ml collection tube.
	4.4	Close the tube gently and centrifuge for 15 seconds at >= 8000 x g.
	4.5	Discard the flow through and collection tube. Transfer the RNeasy column to a fresh 2ml collection tube.
	4.6	Ensure that ethanol has been added to the RPE buffer. Pipette 500ul of RPE buffer onto the Rneasy column.
	4.7	Close tube gently and centrifuge for 15 seconds at >= 8000 x g.
	4.8	Discard the flow through and replace the RNeasy column in the same collection tube.
	4.9	Add another 500ul of RPE buffer to the RNeasy column.
	4.10	Close the tube gently and centrifuge for 2 minutes at >= 8000 x g to dry the RNeasy membrane.
	4.11	Transfer the RNeasy column into a new 1.5ml collection tube. Centrifuge for 1 minute at full speed in a microcentrifuge.
	4.12	To elute, transfer the RNeasy column to a fresh 1.5ml collection tube. Pipette 50ul of RNase free water onto the RNeasy membrane.
	4.13	Close the tube gently and centrifuge for 1 minute at >=8000 x g.
	4.14	Add a second 50ul volume of RNase free water to the column and centrifuge for a further minute at >=8000 x g.
	4.15	The eluted RNA sample is ready for storage. At this stage Rnase inhibitor SUPERase-IN (Ambion #2694) can be added if required. Add 1ul SUPERase-IN per 30ul of RNA. Store at -80ºC.