Apr 8
Summary:
I got 17 samples in total and extracted RNA for all (table 2). Five samples are picked for DNase treatment (table 1).

Table 1:

Sample	Amount of "RNA" before treatment	Amount of RNA after treatment	Percentage of RNA
6D50K	5336	5032	94%
6D50K	4330	3507	81%
6D500K	5298	4141	78%
10D5K	5222	3646	70%
10D200K	5458	3357	61%

In the latter calculation in table 2, I use 60% as the percentage of RNA for all samples.

Table 2:

Sample	Number of larvae (*1000)	Total RNA before DNase treatment (ng)	RNA per 1000 larvea (ng)	60%*(RNA per 1000 larvae) (ng)
6D5K	5	2915.6	583.12	349.872
6D10K	10	1460.3	146.03	87.618
6D25K	25	20639	825.56	495.336
6D50K	50	84194	1683.88	1010.328
6D100K	100	141441	1414.41	848.646
6D500K	500	636868	1273.736	764.2416
6D5K in RNAlater	5	1791.1	358.22	214.932
6D10K in RNAlater	10	3774.3	377.43	226.458
6D25K in RNAlater	25	21141	845.64	507.384
6D50K in RNAlater	50	46551	931.02	558.612
6D100K in RNAlater	100	39741	397.41	238.446
10D5K	5	37442.5	7488.5	4493.1
10D10K	10	102907	10290.7	6174.42
10D25K	25	157248	6289.92	3773.952
10D50K	50	243763	4875.26	2925.156
10D100K	100	532029	5320.29	3192.174
10D200K	200	1569216	7846.08	4707.648

1. I extracted 3 batch of samples, the first and the second batch are not quite consistent, but when I get the hang of the handling, the third batch (the 6 samples in bold) is more consistent and I would trust those data more.
2. I couldn't make a graph of the data because of the huge errors. Most of the errors came from 1) the numbers of larvae in each sample aren't accurate, especially for the samples that contain a small number of larvae, and 2) RNA extraction wasn't consistent for the first two batch.
3. The fourth column in table 2 is the the amount of "RNA" before DNase treatment, and the fifth column is the calculated amount of RNA assuming that 60% are RNA, which is quite conservative based on table 1.

Conclusion:
1. For 6-day-old larvae, 1000 larvae could yield enough RNA for transcription profiling using NGS technologies. For 10-day-old larvae, several hundred would be enough. Those are small numbers and we don't need to worry about not having enough larvae for extraction.
2. Collecting samples using RNAlater would not make much of a difference comparing to dry ice.

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Apr 5, 2010
DNase treatment
Result:

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Apr 2, 2010
DNase treatment
Result: (after and before DNase treatment)

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Apr 1, 2010
RNA extraction
Results:

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Mar 31, 2010
RNA extraction for 11 larvae samples
Results:

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Mar 30, 2010
RNA extraction for WB14 & WB15
Results:

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RNA extraction;
1. Homogenization

• cut tissue (50-100mg)
• add 0.5ml TRI REAGENT
• homogenize tissue samples.
• add another 0.5ml TRI REAGENT
• store at RT for 5min

2. Phase separation

• add 0.2ml chloroform
• vortex vigorously for 15s
• store at RT for 2-15min
• centrifuge at 12,000g for 15min at 4 C

3. RNA precipitation

• transfer the aqueous phase to a new tube
• add 0.5ml isopropanol, mix
• store at RT for 5-10min
• centrifuge at 12,000g for 8min at 4 C

4. RNA wash

• remove the supernatant
• add 1ml 75% ethanol
• vortex to wash the pellet
• centrifuge at 7,500g for 5min at 4 C

5. RNA solubilization

• remove ethanol
• centrifuge for 1min
• remove remaining ethanol
• add DEPC-water