Chao's+Notebook

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

Table 1: In the latter calculation in table 2, I use 60% as the percentage of RNA for all samples.
 * = 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% ||

Table 2: larvae (*1000) || Total RNA before DNase treatment (ng) || RNA per 1000 larvea (ng) || 60%*(RNA per 1000 larvae) (ng) ||
 * Sample || Number of
 * 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.

DNase treatment Result:
 * Apr 5, 2010**

DNase treatment __Result: (after and before DNase treatment)__
 * Apr 2, 2010**

RNA extraction __Results:__
 * Apr 1, 2010**

RNA extraction for 11 larvae samples __Results:__
 * Mar 31, 2010**

RNA extraction for WB14 & WB15 __Results:__
 * Mar 30, 2010**

__RNA extraction__; 1. Homogenization 2. Phase separation 3. RNA precipitation 4. RNA wash 5. RNA solubilization
 * cut tissue (50-100mg)
 * add 0.5ml TRI REAGENT
 * homogenize tissue samples.
 * add another 0.5ml TRI REAGENT
 * store at RT for 5min
 * add 0.2ml chloroform
 * vortex vigorously for 15s
 * store at RT for 2-15min
 * centrifuge at 12,000g for 15min at 4 C
 * 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
 * remove the supernatant
 * add 1ml 75% ethanol
 * vortex to wash the pellet
 * centrifuge at 7,500g for 5min at 4 C
 * remove ethanol
 * centrifuge for 1min
 * remove remaining ethanol
 * add DEPC-water