Affymetrix GeneChip Murine Genome U74Av2 [MG_U74Av2]

A genome-wide study of gene activity reveals developmental signalling pathways active before implantation

rawData

The goal of the experiments was to profile and analyze gene activity during murine pre-implantation development.  Samples were collected at twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst.

DevelopmentalStage

Transcription profiling of mouse pre-implantation development over twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst

The goal of the experiments was to profile and analyze gene activity during murine pre-implantation development.  Samples were collected at twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst.


Affymetrix:Protocol:Hybridization-EukGE-WS2v3[]

Affymetrix:Protocol:Percentile

Title: Affymetrix CEL Analysis (Percentile). Description:

Affymetrix:Protocol:Treatment-Unknown

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-2507

aRNA amplification from 10ng total RNA:  Tian Wang  (Also see Wang et al., Biotechniques 2002 Oct; 33:788)  Day 1:  -Combine 10ng total RNA with 10ng T7-(dT) primer, mix and speed-vac until volume is less then 1ul; adjust volume to 1ul with DEPC-ddH2O;  -Denature RNA and primer at 70 degree for 4 minutes in a thermal cycler with a heated lid; snap cool on ice and keep on ice;  -Add 1ul of ice-cold RT premix to each 1ul sample+primer; mix by pipetting; RT Premix:  (per 5ul)  2.0ul 5X 1st Strand Buffer     1.0ul 100mM DTT     0.2ul 25mM dNTP     0.8ul T4gp32 (5.0mg/ml)     0.5ul RNase inhibitor     0.5ul SuperScript II;  -Incubate for 1hr at 42 degree in a thermal cycler with a heated lid;   -Heat inactivate the rxn at 65 degree for 15 min.; chill on ice;  -Add 13ul ice-cold Second Strand Synthesis (SSS) premix and mix by pipetting; SSS Premix:  (per 65ul)  15ul 5X 2nd Strand Buffer       1.5ul 10mM dNTP       2.0ul DNA Polymerase I (10U/ul)       0.5ul E.coli DNA ligase (10u/ul)       0.5ul RNase H (2U/ul)       45.5ul DEPC-ddH2O;  -Incubate at 16 degree for 2 hours in a thermal cycler;  -Add 0.4ul (2U) T4 DNA polymerase and mix by gentle vortexing; incubate at 16 degree for 15min. in a thermal cycler;  -Heat inactivate the rxn at 70 degree for 10min.;  -Add 55ul TE to bring the volume up to 70ul; add 70ul phenol:chloroform:isoamyl alcohol and mix by pipetting vigorously;  -Transfer the cDNA-phenol mixture to prespun PLG Heavy 0.5ml tubes and spin 5min at 13K rpm;  -Transfer the aqueous phase (~75ul) to a prepared BioGel P-6 column; spin at 1000g for 4 min. recovering the flow-through in a clean 1.5ml eppendorf tube;  -Add 8.5ul 3M NaOAC, 2ul GenElute LPA, 300ul cold ethanol, precipitate at -20 degree overnight;   Day 2:  -Collect cDNA/LPA pellet and wash twice with 50ul cold 80% ethanol;  -Redissolve the pellet in 19ul IVT premix; incubate in 42 degree water bath for 15 min.; pulse spin;   IVT Premix (prepare and hold at room temperature to avoid precipitate): (per 19.0ul)  2.0ul 10X Ampliscribe Buffer         1.5ul 100mM ATP         1.5ul 100mM CTP         1.5ul 100mM GTP         1.5ul 100mM UTP                2.0ul 100mM DTT         0.8ul RNase inhibitor         8.2ul RNase-free ddH2O;        -Transfer each 19ul premix to a PCR tube containing 1.0ul Promega HC T7 RNA polymerase (80U/ul);  -Mix and incubate at 42 degree for 9 hours;  -Purify the cRNA with RNeasy columns; elute with 50ul ddH2O; save 5ul (10%) for electrophoresis analysis and freeze the rest at -20 degree;  -Check recovery on natural agarose gel*;  *samples are SYBR Gold stained; make SYBR loading buffer by adding 1ul SYBR Gold dye to 1ml of 2x denaturing loading buffer; denature RNA by incubating the RNA-loading buffer mixture at 65 degree for 5 min. before loading.  Day 3:  -To each purified cRNA, add 1ul 0.5ug/ul random primer, speed-vac until the volume is less than 5ul;  -Heat the aRNA/primer mixture to 70 degree for 5 min. in a thermal cycler with a heated lid; snap cool on ice and keep at room temperature for 10 min.;  -Add 5ul room temperature RT premix and mix by pipetting; RT Premix:  (per 25ul)    10.0ul 5X 1st Strand Buffer          5.0ul 100mM DTT          1.0ul 25mM dNTP          4.0ul T4gp32 (5.0mg/ml)          2.5ul RNase inhibitor          2.5ul SuperScript II;  -Adjust volumes to 10ul per rxn; incubate in a thermal cycler according to the following:   37 degree 20min   42 degree 20min   50 degree 10min   55 degree 10min   65 degree 15min   hold at 37 degree;  -Add 0.5ul (2U/ul) RNase H, mix by gentle vortexing and incubate at 37 degree for 30min.;  -Heat to 95 degree for 2 min., chill on ice and keep on ice;  -Add 1ul ice-cold 100ng/ul T7-(dT) primer; incubate at 70 degree for 5 min. and then 42 degree for 10 min. to anneal the primers;  -Prepare SSS Premix minus ligase; Round 2 SSS Premix : (per 63.5ul) 15.0ul 5X 2nd Strand Buffer               1.5ul 10mM dNTP        2.0ul DNA Polymerase I (10U/ul)               0.5ul RNase H (2U/ul)              44.5ul DEPC-ddH2O;  -Snap cool samples on ice and add 63.5ul ice-cold SSS Premix for a 75ul SSS rxn volume; incubate at 16 degree for 2 hours;  -Add 2ul (10U) T4 DNA polymerase and mix by gentle vortexing; incubate at 16 degree for 15min.;  -Heat inactivate the rxn at 70 degree for 10min.;  -Extract with 75ul phenol in PLG Heavy 0.5ml tubes;   -Transfer the aqueous phase to prepared BioGel P-6 columns, spin at 1,000g for 4 min. to recover the flow-through;  -Use 5ul of the flow-through for electrophoresis analysis (also use SYBR Gold, add 1ul SYBR Gold to 1ml of 6x DNA loading buffer);  -Precipitate the rest flow-through at -20 degree O/N with 9ul 3M NaOAC, 2ul GenElute LPA and 300ul cold ethanol;   Day 4:  -Collect the cDNA/LPA pellet and wash twice with 250ul 80% ethanol;  -Redissolve the pellet in 11ul DEPC-ddH2O;  -To the cDNA, add the following IVT labeling mix;  IVT labeling mix (Enzo):  (per 20ul rxn) 11ul  cDNA      2ul 10x HY Reaction Buffer      2ul 10x Biotin-labeled NTP Mix      2ul 10x DTT      2ul 10x RNase Inhibitor      1ul  Promega HC T7 RNA Polymerase (or Enzo enzyme) (80U/ul);  -Mix and pulse spin (5 sec.); immediately incubate at 42 degree for 5 hours in a thermal cycler;  -Purify the aRNA with RNeasy Mini Columns; elute with 35ul RNase-free ddH2O;  -Use 2ul of elute to quantify by OD (dilute 1:40 in TE); set up fragmentation rxn with 32ul of elute; save the rest 1ul for electrophoresis analysis;  Fragmentation rxn:  (per 40ul) 32ul elute from RNeasy Columns      8ul 5x Fragmentation Buffer    -----------------------------------------    35min. at 94 degree; chill on ice and keep on ice;  -Use 1ul of the fragmentation mixture for electrophoresis analysis on EtBr gel;  -Set up hybridization cocktail with 10ug aRNA; set up hybridization according to Affymetrix;    Reagents:  Obtained/made per instruction in Affymetrix GeneChip Expression Analysis Technical Manual:  T7-(dT) primer, Enzo labeling kit, fragmentation buffer;  From Invitrogen: everything in cDNA synthesis except T4 gene 32 protein (from USB);  From Epicentre Technologies: Ampliscribe T7 transcription kit;  From Qiagen: RNeasy columns;  From Biorad: BioGel P-6 columns;  From Molecular Probes: SYBR Gold dye;  From Fermentas: 2x RNA denaturing loading dye;  From Eppendorf: Phase Lock Gel tubes;  From Sigma: linear polyacrylamide (LPA). 

Transcription profiling of mouse pre-implantation development over twelve time points from the germinal vesicle (GV) stage oocyte to the late (expanded) blastocyst

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Samples