-- -RNA Sample Processing:
- -Trizol RNA isolation and RNeasy clean up protocol for whole plants (embryo-derived tissue dissected from 4 days old germinating grains) and the seedling leaves (12 days after planting).
- -- -
-☠Grind tissue (9 embryos) with a mortar and pestle in liquid nitrogen
-☠Add 5 ml TRIzol (pre-heated to 60oC) to all samples, vortex until all the tissue is thawed, place in the 60oC waterbath..
-☠Incubate samples at 60oC for 10 minutes, vortexing three times.
-☠Centrifuge @ 4000 x rpm @ 4C for 30 minutes (in Eppendorf 5810R).
-☠While centrifuging, label new set of 15 ml tubes
-☠Transfer supernatant to 15 ml centrifuge tube
-☠Add 1 ml of chloroform. Vortex the sample until color shade is uniform at least 5
-seconds, and incubate at room temperature for 5 minutes.
-☠Centrifuge @ 4000 x rpm for 30 minutes @ 4oC.
-☠While centrifuging, label new 15 ml tubes
-☠Collect the upper aqueous layer (there will be about 3 mls) and transfer to a new 15 ml tube.
-☠Add 0.6 volumes (2 ml) of isopropanol, mix gently, incubate at room temperature for 20 minutes.
-☠Centrifuge @ 4000 rpm for 30 minutes @ 4oC.
-☠Wash the pellet with 10 ml of cold 75% ethanol. Swirl & centrifuge at
-4000 rpm for 15 minutes @ 4oC.
-☠Discard supernatant, centrifuge for 5 min, remove the rest of the ethanol
-☠Air-dry the pellet for 10 minutes, inverted on a kimwipe.
-☠Dissolve pellet in 400 ul of DEPC-treated H2O. Resuspend by pipeting up & down a
-few times.
-☠Add 2 ul SuperaseIn. Incubate at 60oC for 10 minutes to resuspend.
-☠Set water bath to 37oC.
-☠Add 50 ul 10X DnaseI Buffer, 45 ul H2O and 5 ul of DnaseI, incubate at 37oC for 1 hr.
-☠Prepare Buffer RLT (Rneasy Clean-up Midi Kit) by adding b-mercaptoethanol (10ul/1ml RLT).
-☠Add 2.0 ml Buffer RLT to the RNA prep and mix thoroughly.
-☠Add 1.4 ml ethanol (96-100%) to the diluted RNA. Mix thoroughly.
-☠Label 15 ml tubes from the kit and place midi columns in them
-☠Apply sample to a Midi column, close tube gently and centrifuge for 20 min at 3000 rpm.
-☠Discard the flow-through.
-☠Add 2.5 ml Buffer RPE to the RNA easy column, close the centrifuge tube gently,
-incubate for 3 min
-☠Centrifuge for 10 min at 3000 rpm. Discard the flow-through.
-☠Add another 2.5 ml Buffer RPE to the RNeasy column. Close the centrifuge tube
-gently, incubate for 3 min
-☠Centrifuge for 10 min at 3000 rpm, remove flow-through
-☠Centrifuge again for another 5 min.
-☠Label new 15 ml tubes from the kit.
-☠Transfer the RNA easy column to a new tube and pipet 250 ul volume of
-RNase-free water directly onto the RNeasy silica-membrane incubate for 1 min
-☠Centrifuge for 5 min at 3000 rpm.
-☠To the same tube add again 250 ul H2O, incubate for 1 min.
-☠Centrifuge for 5 min at 3000 rpm.
-☠Label two sets of 1.5 ml Eppendorf tubes.
-☠Transfer 490 ul to the one tube and 10 ul to another one. Use 10 ul tube for the RNADetailed descriptions of these procedures can be found under the ArrayExpress (http://www.ebi.ac.uk/aerep/?) protocol P-MEXP-4631 (Caldo et al. 2004).
- -Replication and Sample Balance:
- -3 independent replicates of both parental cultivars Steptoe and Morex were generated for both tissues, embryo and seedling leaf.
- -Experimental Design and Batch Structure:
-
--- cgit v1.2.3The following are ArrayExpress (http://www.ebi.ac.uk/aerep/?) experiment IDs: E-TABM-111 (leaf, 41 chips) and E-TABM-112 (embryo derived, 156 chips).
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