Embryo culture and single blastomere isolation
For most experiments, frozen late 2-cell stage embryos (B6C3F1 females bred with B6D2F1 males) were obtained from Embryotech Laboratories, Inc. (Wilmington, MA), and were cultured as previously described  until the desired stage of development. For the DNase experiment, frozen 8-cell embryos obtained from the same source were grown to the morula stage. Blastocyst stage embryos used for hsp70i measurements were also grown from frozen 8-cell embryos.
Single blastomeres were isolated from either 4-cell embryos (for Xist measurements) or pre-compaction 8-cell embryos (for hsp70i measurements) after drilling the zona using a ZILOS-tk™ zona infrared laser optical system (beam = 1480 nm) (Hamilton Thorne Biosciences, Inc., Beverly, MA), according to a procedure developed in our laboratory and described elsewhere [18, 19].
PurAmp multiplex measurements of Xist/SryRNA + DNA in individual embryos or blastomeres
All experimental procedures were carried out using rigorous precautions aimed at avoiding or destroying environmental RNases contamination [17–19].
Dried droplets of denaturing solution, hereafter called "LysoDots", were prepared prior to embryo collection by delivering 20-nl aliquots of the denaturing solution (see below) to the inside surface of the lids of PCR-grade reaction tubes (Applied Biosystems, Foster City, CA). Precise measurement of the droplets size was obtained following the method previously described by Wangh . The denaturing solution composition was: 0.25% sarcosyl, 2 M GITC, 100 mM β-mercapto-ethanol, 0.01 M sodium citrate, pH 7.0 (all reagents from Stratagene, La Jolla, CA), 1% (vol/vol) dimethylsulfoxide (Sigma Chemical Company, St. Louis, MO). LysoDots were prepared in advance, allowed to dry under sterile conditions, and then stored at room temperature in closed PCR tubes.
Immediately before harvesting, individual embryos were placed in 3 ml of Dulbecco's PBS devoid of calcium and magnesium chloride . Dulbecco's PBS containing 0.4% polyvinyl pyrrolidone (both products from Sigma) was used when isolating single blastomeres . After one wash in the same buffer, each embryo or cell was aspirated into a glass capillary having an internal diameter of 0.2 mm  and tapered at the end so that the inner volume of the tapered tip would contain about 20 nl. Tapering was obtained by pulling the glass capillaries in a Micro-Pipette Puller (Industrial Science Associates, Inc., Ridgewood, NY). The embryo (or cell) was expelled directly onto the LysoDot in a volume of PBS as close as possible to 20 nl. Microscope observation revealed that the GITC crystals dissolved instantly upon addition of the sample-containing PBS and, thus, that cell lysis occurred immediately. Tubes were closed upside down and heated at 75–77°C for 5 minutes, after which their content was once again dry or semi-dry. The samples were then stored at -20°C until the next step.
In order to perform reverse transcription, each sample was carefully re-solubilized in the lid by addition of 6 μl of Random Hexamers mixture (4.2 ng/μl) in DEPC-treated water (all RT reagents were from a ThermoScript™ RT-PCR System kit, Invitrogen, Life Technologies, Carlsbad, CA). Tubes were closed, inverted, briefly centrifuged and incubated for 5 minutes at 65°C in order to allow primer/RNA hybridization. The remaining reagents needed for RT were then added to the tube in a volume of 4 μl, and the reaction was carried out according to the protocol suggested by the manufacturer. As previously described [17–19], all RT reagents were used at the suggested concentrations except for the absence of DTT, but volumes were halved so that each assay was performed in just 10 μl, which increased to 10.5 μl after RNase H digestion.
The full volume of each sample was then mixed with 89.5 μl of complete PCR amplification cocktail containing sequence-specific molecular beacons as detection probes . Multiplex real-time PCR of Xist and Sry genomic DNA + cDNA templates was thus performed in a final volume of 100 μl, as detailed elsewhere, in the presence of 4 units of Taq DNA polymerase (Promega, Madison, WI) [18, 19]. Real-time PCR was carried out in an ABI Prism® 7700 Sequence Detector (Applied Biosystems, Foster City, CA) and fluorescence readings were taken at the annealing temperature.
PurAmp assay for hsp70iRNA and DNA measurements in individual embryos or blastomeres
Embryos were heat-shocked at 43°C for 30 minutes, followed by a recovery period of 30–40 minutes (blastocysts), or 2–3 hours (8-cell embryos, as indicated) at 37°C. The hsp70i assay was carried out similarly to the one for the Xist/Sry multiplex, by sequential dilutions of denaturant, RT and PCR reagents. The procedure for collection and lysis of the samples was the same. Dry samples were re-solubilized with 6 μl of random decamer primers (8.3 μM) in nuclease-free water (all RT reagents were from a Cells-to-cDNA™ II kit, Ambion, Inc., Austin, TX). After a 3 minute incubation at 75°C to optimize primer binding to RNA, all other reagents needed for RT were added to the sample and the reaction was carried out according to the manufacturer's instructions. As for the Xist/Sry assay, all RT mixture components were used at the suggested concentrations, but volumes were halved so that RT was performed in a final volume of 10 μl. An RNase H digestion step was included at the end of RT, as in the case of the Xist/Sry assay.
Real-time PCR was carried out in a final volume of 100 μl, by adding the PCR reagents to the sample after completion of RNase H digestion. The chosen hsp70i primers were localized at positions 1245/1305 of the hsp70.1 GenBank sequence with accession number M35021 (5' CCGCCTACTTCAACGAC 3', upstream primer; 5' ATCCGCAGCACGTTTA 3', downstream primer) and were identical to sequences within the hsp70.3 gene, previously known as hsp70A1 (GenBank sequence with accession number M76613) . Because the hsp70i was the only amplicon generated in this assay, it was not necessary to design a sequence-specific detection probe. In this case, SYBR® Green, a fluorescent dye that binds to double-stranded DNA, was used as fluorescent probe for real-time PCR. The specificity and purity of the amplicon was confirmed by both gel electrophoresis and analysis of the melt profile, as previously described . The composition of the cocktail for hsp70i PCR was the following: 50 mM Tris, pH 8.3, 3 mM MgCl2, 0.3 μM each primer, 0.25 mM each dNTP, 1:62,500 SYBR Green (from a "10,000X concentrate in DMSO" purchased from FMC BioProducts, Rockland, ME), and 4 units of Taq DNA polymerase (Promega, Madison, WI). The polymerase was incubated at a 1:1 (v/v) ratio with Platinum® Taq antibody (Invitrogen) for 5 minutes before addition to the reaction mixture (hotstart PCR). The cycling profile was: 95°C for 5 minutes; 10 cycles consisting of the following four steps: 95°C (20 sec), 64°C (30 sec), 72°C (30 sec), 84°C (15 sec); 35 cycles with the following four steps: 95°C (20 sec), 59°C (30 sec), 72°C (30 sec), 84°C (15 sec). Fluorescence readings were acquired at 84°C, in order to exclude fluorescent signals due to the possible formation of primer dimers late in the reaction.
A number of embryos were also processed as "No RT" controls, with the same protocol used for the other samples but without inclusion of reverse transcriptase in the RT mixture. These controls were used to quantify hsp70i genomic DNA copy numbers in the absence of cDNA .
Multistep/multitube nucleic acid extraction
For the preliminary studies on hsp70i expression in blastocysts, some of the embryos were processed using a commercially available multistep/multitube kit, as previously described . Briefly, nucleic acids (DNA and RNA) from each sample were purified using phenol:chloroform:isoamyl alcohol phase separation (Micro RNA Isolation Kit, Stratagene, La Jolla, CA) with a ratio of 100 μl of phenol and 45 μl of chloroform/isoamyl alcohol solution per assay. Transfer RNA (10 μg/assay; Sigma Chemical Company, St. Louis, MO) was added as a co-precipitant. Pellets were washed twice, once in isopropanol followed by overnight precipitation at -20°C and once in 75% ethanol, and then nucleic acids were reverse transcribed and analyzed by real-time PCR exactly as detailed above for the PurAmp-treated samples.
Quantification of Xist, Sry and hsp70iamplicons
Calculation of template copy numbers was based on the "threshold cycle" (CT) at which each fluorescent signal was first detected above background. CT values were compared to standard scales obtained from analysis of male mouse genomes at known copy numbers, as detailed previously [17–19] and further exemplified in the Result section. Briefly, a two-fold difference in the number of templates amplified results in a shift of one cycle between two CT determinations. A lower CT value indicates an earlier detection of the fluorescent signal and therefore more templates present at the start of the reaction [reviewed in ref. ]. One male mouse genome contains one copy of Sry (Y-chromosome), one copy of Xist (X-chromosome) and four copies of hsp70i (two of hsp70.1 and two of hsp70.3, both on chromosome 17).
Insertion of a DNase I digestion step within the Xist/SryPurAmp protocol
Embryos were grown to the morula stage and were individually harvested and lysed as detailed above. Controls were processed for multiplex detection of Xist/Sry with the described PurAmp protocol. RNA-only samples were prepared by inserting a DNase digestion step prior to RT, as follows. Lysed, dried samples were re-suspended with 4 μl of DNase mixture containing: 20 mM Tris-HCl, pH 8.4; 2 mM MgCl2, 50 mM KCl (Invitrogen's DNase I Reaction Buffer) and 1 unit of DNase I (Ambion) in nuclease-free water. After an incubation of 20 minutes at room temperature, the reaction was terminated by adding 1 μl of a 10 mM EDTA solution, pH 8.0 (Invitrogen). The nuclease was inactivated by heating the samples at 65°C for 10 minutes, according to the protocol recommended by Invitrogen. One μl of a 25 ng/μl RT primer solution was then added to the sample, so that the final primer concentration was now the same as in the assay without DNase (see above). RT and PCR were then carried out as detailed for the "No DNase" assay.