Protocol for cDNA library construction and chip analysis of small amount tissue
2002/11/12
Outline: （aRNA amplification）
1. Dissect primordia
2. Homogenizing
3. Total RNA isolation
4. First strand cDNA synthesis
5. Second strand synthesis
6. ds cDNA cleanup
7. In vitro transcription
8. aRNA cleanup
(two round)
9. aRNA amplification: first strand cDNA synthesis
second strand synthesis
10. Check the amplified antisense RNA on gel.
Flow Chart
1. Dissect primordia:
Add 20ul TRIzol in a 1.5ml Eppendorf tube put on ice
(11/12 2~3hr)
Treatment the homemade dissecting knife and needles with DEPC water
Add several drops DEPC water on the slide
Carefully detached the panicle, put it in the water
Put the slide under dissecting microscope
Carefully separate the needed primordia, put in TRIzol in the Eppendorf tube
2. Homogenizing
(30min)
Homogenizing (NOTE: don’t let the temperature of the tube increased too high, put on ice
intermittently)
Use 20ul/(40ul for 20 primordia) TRIzol to wash the homogenizer once, collect it into the same
tube
3. Total RNA isolation
Add 0.5ul LPA (10ug/ul), mix
(30min+O/N, and 1hr of next day)
Add 10ul/20ul CH3Cl, mix
Rest still at RT (room temperature) for 2min, mix intermittently
Centrifuge 4℃, 12,000rpm, 20min
Aspirate supernatant to a new tube (~20ul/30ul)
Add 40ul/60ul isopropyl alcohol, leave O/N at -20℃
Centrifuge 4℃, 13,000rpm, 25min (prepare 3ml 75% ethanol during this step)
Remove supernant by pipette, wash precipitation with 500ul 75% ethanol
Centrifuge 4℃, 13,000rpm, 5min
Remove supernant by pipette, wash precipitation with 500ul 75% ethanol.
Centrifuge 4℃, 13,000rpm, 5min
Remove supernant by pipette. Pulse-spin, remove remaining sup, air dry
4. First strand cDNA synthesis
Set up first strand cDNA synthesis presolution in a
0.5ml Eppendorf tube as right
(11/13 2hr)
4.5ul
total RNA in DEPC H2O
0.5ul
0.1ug/ul oligo dT(15)-T7 primer
70℃ for 5min, snap cool on ice
Set up first strand cDNA synthesis reaction as right, add
it in above-mentioned presolution, make up the dropped
volume with water
2ul
5×First strand buffer
1ul
0.1M DTT
0.5ul
10mM Ultrapure dNTP
0.5ul
RNaseIN
42℃ 40min
0.5ul
T4gp32 (8.0mg/ml)
50℃ 10min
0.5ul
Superscript II (100U)
55℃ 10min
65℃ 15min
Incubate in a thermal cycler with a heated lid, but not in a water bath.
Chill on ice.
5. Second strand synthesis
Set up second strand synthesis premix as left
Add it into 10ul first strand reaction, and mix by pipetting
(NOTE: be sure the premix is cold when add it)
Incubate at 15°C for 2 hours
(3hr)
45ul
DEPC H2O
15ul
5×Second-Strand Buffer
1.5ul
10mM dNTP mix
0.5ul
RNase H (2U/ul)
1ul
E. coli DNA Ligase (5U)
2ul
DNA Polymerase I (10U)
Add 1ul (10U) T4 DNA Polymerase, and mix by flicking and gentle vortexing
Incubate at 15°C for 15min
Heat inactivate the reaction by heating it to 7°C for 10min
6. ds cDNA cleanup
Add 175µl RNase-free water
(1.5hr, can stop after this step)
Add 250µl phenol-chloroform-isoamyl alcohol 25:24:1 to the sample, vortex vigorously for at
least 30sec.
Spin at max speed for 10min in a tabletop centrifuge
Apply the upper water phase to a YM-100 Microcon column (NOTE: Be careful no to bring along
any phenol to the YM-100 column.)
Spin the YM-100 column at 500×G in a tabletop centrifuge for 10min. (NOTE: The YM-100
columns are not recommended to be used together with DNA samples at forces higher than 500 x
G. After centrifugation, verify that there has been an adequate flow-through of the sample, i.e.
there is less than 50 µl of liquid left in the column.)
Remove the column from the holder tube and discard the liquid in the holder tube
Put the column back into the holder tube
Apply 500µl of RNase-free water to the YM-100 column and spin at 500×G for 10min. (Check
that there is less than 100 µl of liquid left in the column. If not, spin at 500×G for another couple
of minutes.)
(three times)
Discard the flow-through
Elute the Sample from the YM-100 column by placing the column upside down into a collector
tube (provided)
Spin at max speed for 1min
Add 1/25 vol. 5M NaCl and 2.5 vol. Ethanol, mix, leave O/N at -20℃
Centrifuge 4℃, 13,000rpm, 20-30min
Remove supernant by pipette, wash precipitation with 500ul 75% ethanol
Centrifuge 4℃, 13,000rpm, 2-3min
Remove supernant by pipette. Pulse-spin, remove remaining sup, air dry
7. In vitro transcription
Set up in votro transcription reaction in a
0.5ml Eppendorf tube as right, use it to
resolve the recovered RNA above
Add 1.0ul T7 RNA polymerase, mix by
flicking and gentle vortexing
(11/14 6hr)
6ul
25mM NTP Mix (A, G, C and UTP) (if
new kit, combine NTPs into one tube)
2ul
10×Ampliscribe Buffer
2ul
100mM DTT
0.75ul
RNase inhibitor(~30U)
ds cDNA in DEPC H2O
8.25ul
Incubate at 42℃ for 5hr
Freeze or proceed
8. aRNA cleanup
(1hr)
Prepare the GenElute kit lysis buffer by adding 2.3ul β-ME (supplied) per 230ul of lysis buffer.
To the 20µl in vitro transcription reaction, add 230µl of GenElute lysis buffer
Add 250µl 70% ethanol. Mix by vortexing.
Transfer into blue filtration column; spin 15 sec at higher than 14,000g
Add 500ul wash solution 1 to column; spin 15 sec. (The high-salt wash may be necessary to
remove short-length RNA byproducts from the in vitro transcription reaction.)
Add 500ul wash solution 2 (low-salt wash; add 10ml Ethanol per 2.5ml before use) to column;
spin 15 sec.
Add 500ul wash solution 2; spin 2 min.
Transfer column to new collection tube, add 50ul elution solution, spin 1min. (Repeat if >100ug
RNA expected)
Transfer the 50ul sample to a 500µl microtube
Add 1/25 vol. 5M NaCl and 2.5 vol. Ethanol, mix, leave O/N at -20℃
Centrifuge 4℃, 13,000rpm, 20-30min
Remove supernant by pipette, wash precipitation with 500ul 75% ethanol
Centrifuge 4℃, 13,000rpm, 2-3min
Remove supernant by pipette. Pulse-spin, remove remaining sup, air dry
9. aRNA amplification:(two round)
first strand cDNA synthesis
Set up first strand cDNA synthesis presolution in a 0.5ml
Eppendorf tube as left
(11/15 5hr)
4ul
aRNA in RNase-free water
1ul
Random hexamers (50ng/ul)
Heat at 70°C for 5 min, snap cool on ice and sit at room temperature for 5min
Set up first strand cDNA synthesis premix; add it into
above-mentioned presolution.
37°C for 20min
42°C for 20min
50°C for 10min
55°C for 10min
65°C for 15min
hold at 37°C
2ul
5×First strand buffer
1ul
0.1M DTT
0.5ul
10mM Ultrapure dNTP
0.5ul
RNaseIN
0.5ul
T4gp32 (8.0mg/ml)
0.5ul
Superscript II (100U)
Add 1U RnaseH, and mix by vortexing gently
Incubate for 30min at 3°C and then heat to 9°C for 2min
Chill on ice and then spin briefly to collect condensation and return to ice
second strand synthesis
Add 100ng (dT)-T7 (as 1µl) while on ice
Incubate at 4°C for 10 min to anneal the primer.
Set up second strand synthesis premix as left
Add it into 10ul first strand reaction, and mix
by pipetting (NOTE: be sure the premix is cold
when add it)
45ul
DEPC H2O
15ul
5×Second-Strand Buffer (Life Tech)
1.5ul
10mM dNTP mix
0.5ul
RNase H (2U/ul)
2ul
DNA Polymerase I (10U)
Incubate at 15°C for 2 hours
Add 2ul (10U) T4 DNA Polymerase, and mix by flicking and gentle vortexing
Incubate at 15°C for 15min
Heat inactivate the reaction by heating it to 7°C for 10min
(Second round
amplification)
ds cDNA cleanup
In vitro transcription
aRNA cleanup
(1.5hr to O/N)
(11/16 7hr)
(1.5hr)
10. Check the amplified antisense RNA on gel.
(11/18 2hr)
Use 5ul to check on gel, dilute 6ul into 100ul to check with spectrophotometer, 9ul to first strand
synthesis (20ul system), others same as first round.
左图显示 RNA 的 28s 及 18s，18s 接近 2kb，5s 大约为 20bp 左右，所得的 aRNA 大致在 18s
及 5s 之间。
分光光度值：A260=0.153; A280=0.083; A260/A280=1.846
1OD（A260）表示每毫升含 40ug RNA，可计算出 20ul 中约含 2.4ug aRNA。
T7 aRNA 扩增需购试剂
第一链合成试剂：
1 T7 Primer: 5'-GCA TTA GCG GCC GCG AAA TTA ATA CGA CTC ACT ATA
GGG AGA (T)15 VN-3' (V = A, C, and G, N = A, T,G,C), 59mer
2
10 mM dNTP (Pharmacia, cat#27-2035-02)
3 TS Primer: 5’-AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG-3’, 30mer
4 T4gp32 (USB, cat#70032Y 100UG)
5 Superscript II (已购)
第二链合成试剂：
1 Second strand synthesis (Invitrogen): DNA Polymerase I (Cat#18010-017) E. coli
DNA Ligase (Cat#18052-019) T4 DNA Polymerase (Cat#18005-025) RNase H
(Cat#18021-014) 5X second strand buffer (Cat#10812-014)
2 Advantage polymerase kit (clontech Cat#8417-1)
说明：综合两个实验室 protocol 的试剂，满足所有 cDNA 合成方法，也可用 PCR 扩增检测
纯化：
1
2
YM-100 Microcon columes(Millipore, Cat#42424)
Genelute Mammalian total RNA miniprep kit (sigma, Cat#RTN-10)
转录：
Epicentre ampliscribe kit (Epicentre, Cat#AS2607)
第二次第一链合成：
random primer (promega, Cat#C1181)
说明：只用于转录产物的反转录
试剂 LPA 自配
品名：微量组织匀浆器 Micro Tissue Grinders
厂家：KIMBLE KONTES
货号：749521-0590
说明：槌 0.5ml 一次性