13 January 2014

FastAnnotator and RNA extraction

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Monday notes

ApTranscriptome

FastAnnotator results are in!

ApGxL

Bioanalyzer results for last week’s extractions:

RIN (RNA Integrity) values

Sample	RNAzol ng/ul	RNAzol 260/280	RNeasy ng/ul	RNeasy 260/280	BioAn ng/ul	BioAn RIN
A22-00	-	-	9.97	1.78	6.7	7.5
A22-07	-	-	31.78	1.93	20.6	6.7
A22-10	-	-	6.99	1.84	3.5	8.0
04-A HS	23.06	1.69	17.12	1.54	12.3	7.2
04-B HS	12.89	1.81	11.27	1.42	4.8	7.0
05-B HS	63.73	1.66	14.48	1.82	9.3	7.0
06-A HS	80.00	1.69	11.19	1.46	3.4	6.8
06-B HS	31.24	1.70	5.68	1.15	0.8	7.5
08-A HS	47.97	1.65	8.32	1.55	2.1	7.9
09-A HS	22.68	1.60	5.31	1.11	0.2	7.6
10-A HS	42.40	1.59	9.11	1.35	1.0	7.2
10-B HS	13.10	1.61	15.18	1.34	0.6	6.9
12-A HS	38.57	1.69	14.38	1.52	4.0	7.8
12-D HS	21.5	1.57	14.85	1.33	0.2	8.4
13-A HS	17.44	1.63	9.24	1.24	-	-
13-B HS	10.58	1.49	9.34	1.30	0.9	7.8
16-A HS	6.31	1.57	9.76	1.45	0.7	7.6
16-B HS	15.26	1.59	5.55	1.00	0.2	6.8
21-A HS	19.00	1.64	8.97	1.29	0.6	8.0
21-B HS	16.85	1.58	4.83	1.21	0.06	4.6
22-A HS	13.55	1.38	5.73	1.19	0.3	8.5
22-B HS	22.9	1.67	11.35	1.35	0.2	8.2
22-C HS	66.41	1.66	21.77	1.62	11.4	7.6

First three samples (A22-00, A22-07, A22-10) are from last Tuesday’s trial extraction.

• Bioanalyzer concentration (ng/ul) are all below Nanodrop…in some cases considerably
• with 25 ul elution volume, need 4 ng/ul to get 100 ng. only 7 of 23 reach this and one of these (A22-07) has RIN below 7

---

RNA extraction

• 12 samples
• only did one ethanol wash for RNAzol
• eluted in 54 ul after RNAzol
• took 2 ul aliqout for Bioanalyzer
• proceeded with RNeasy as before

Sample	RNAzol	RNAzol	RNeasy	RNeasy
01-C HS	1281.5	1.8	11.7	1.42
02-A HS	571.9	2.33	6.28	1.33
02-C HS	174.7	1.77	3.29	1.29
10-C HS	120.6	1.72	4.35	1.3
11-A HS	259.3	1.95	0.2	-0.23
11-B HS	191.11	1.82	1.8	2.8
12-A HS	395.6	2.11	7.2	0.35
13-A HS	209.7	1.84	9.75	1.51
13-C HS	129	1.67	13.48	1.4
15-A HS	126.3	1.69	9.5	1.46
15-C HS	269.2	1.9	6.26	1.5
16-A HS	463.2	2.2	2.99	1.55

Disappointing - massive loss in yield from RNAzol sample after RNeasy. Probably because starting with small amount of biological material.

Check Bioanalyzer results - if high quality RIN values after RNAzol, use this step only.

Otherwise - return to using 3 ants per extraction (instead of 1) as this yielded good results before for transcriptome assembly.

CCThermBe

Set up first foster colony with about 50 pupae with Grace.