The PurAmp method presented in this study is performed in a single tube from cell lysis to cDNA or genomic DNA amplification, thus eliminating possible loss of template molecules due to procedures such as phase separation and recovery, repeated washing and re-suspension of nucleic acid pellets, elution from binding matrices and vessel-to-vessel transfer. This strategy offers an immediate improvement in the precision of gene expression analyses, at the same time shortening considerably the experimental protocol compared to traditional methods.

In order to validate our method, we initially measured the Xist RNA content of a group of female mouse embryos at the blastocysts stage and quantified Xist and Sry DNA copy numbers in their male counterparts. Our previous analysis of these parameters in single female and male mouse embryos at different developmental stages provided us with an ample pool of data obtained with commercially available nucleic acids preparation methods 171819, which we used as a reference for comparison with our new results.

Figure 1 shows the real-time PCR plots obtained from six PurAmp-treated single embryos. Following RT, the accumulation of multiplexed Xist/Sry amplicons was detected using two molecular beacons conjugated to different fluorescent dyes. Both amplicons span intronless sequences of the genes 18, thus allowing in either case the simultaneous measurement of cDNA (when present) and genomic DNA copies. Each color in the plots of Fig. 1 identifies a specific embryo and is used for both its Sry and Xist signal. Three embryos were identified as male based on the presence of the Sry amplicon (Fig. 1, upper panel, lines in blue hues) and on the fact that in each case the Xist fluorescent signal (Fig. 1, lower panel, lines in blue hues) arose at the same "threshold cycle" (C_T) as the Sry signal (see Methods for a definition of C_T and details on signal quantification). This indicates the presence of the same number of copies of Sry and Xist templates, as expected for male blastomeres that contain one copy of the Sry gene on the Y-chromosome and one copy of the Xist gene on the X-chromosome. Neither gene is expressed in male blastocysts 1617, and therefore these three samples contained only genomic Xist and Sry DNA. The three remaining embryos did not generate any Sry signal (Fig. 1, upper panel, lines in red hues), while their Xist signals arose significantly earlier than the others (Fig. 1, lower panel, compare lines in red and blue hues). Based on these data they could be identified as female embryos which contained thousands of copies of Xist RNA in addition to two copies of genomic Xist DNA per cell. These results are fully in agreement with those of our earlier analyses using traditional methods of nucleic acid preparation.

Quantification of the real-time PCR data obtained from the three male embryos in Fig. 1 indicated that, on average, each embryo contained 125 ± 83 (mean ± s.d.) copies of Xist genomic DNA, consistent with the previous estimate of 165 ± 101 genomes per male blastocyst 17. Both values are higher than the expected cell number per embryo at this stage (60–100, depending on culture conditions), due to endoreduplication in trophoblast cells 21, a phenomenon also responsible for some variability between samples. In contrast, a total of five female blastocysts analyzed via the PurAmp method yielded an average of 12,600 ± 5079 copies of Xist cDNA + genomic DNA per embryo. Since each female blastocyst contains about 250 copies of the Xist genomic sequence (twice the number of a male embryo), the accumulation of Xist RNA per female blastocyst averages above 12,000 copies, considerably higher than our previous measurement of 6797 ± 2894 copies obtained using a multistep nucleic acids isolation procedure 17. The data in Figure 2 demonstrate that the amplification efficiency of both the Xist and Sry sequences is neither decreased nor increased by the presence of diluted denaturing solution (0.4 mM GITC, as in the PurAmp protocol) during real-time PCR. In fact, additional experiments revealed that Xist/Sry real-time PCR was unaffected by a GITC concentration as high as 20 mM (not shown). Taken together these results suggest that the higher levels of Xist cDNA measured using the PurAmp method are due to an improvement in RNA recovery at the initial step of cell lysis.

As shown above, blastocysts are comprised by many cells and contain hundreds of copies of the Xist and Sry genes and thousands of copies of Xist transcripts, with rather wide sample-to-sample fluctuations. In order to more carefully determine the quantitative capability of the new assay, we next analyzed embryos at earlier developmental stages containing lower and, in some cases, precisely known numbers of template copies. Figure 3 illustrates the real-time PCR plots of the Xist amplicons generated in the course of two separate experiments by, right-to-left, i) a 3-cell male embryo (yellow); ii) a 4-cell male embryo (green); iii) a single blastomere isolated from a 4-cell female embryo (light purple); iiii) a 4-cell female embryo (red); iiiii) a female blastocyst (blue). The gender of each embryo was confirmed by the detection of an Sry-specific fluorescent signal in male samples (Fig. 3, inset). The quantitative analysis of these results confirmed that the 3-cell male embryo contained 3 copies of the Xist gene, while the 4-cell male embryo contained 6 copies of the Xist gene, indicating that DNA duplication had occurred in two of the blastomeres. It has long been known that two of the blastomeres of a 4-cell embryo divide ahead of the other two 22, an observation in agreement with our finding. The numbers of Xist templates measured in these male embryos also confirmed the expectation that these samples did not contain Xist RNA because Xist is not expressed in male cells 1317. Conversely, the Xist signal of the female 4-cell embryo arose about five cycles earlier than the Xist signal of the 4-cell male embryo (compare red and green curves), denoting the presence of Xist transcripts (157 copies of Xist RNA assuming diploidy of all cells and calculated from a total of 165 cDNA + genomic DNA templates), albeit at considerably lower levels than those measured in the female blastocyst (9750 copies of Xist RNA assuming the aforementioned average number of genomes per blastocyst of 125, and based on 10,000 copies of total Xist cDNA + genomic DNA templates). These measurements are consistent with other studies demonstrating that Xist transcripts are accumulated in the developing embryo beginning at the late 2-cell stage 23 and with our previously published Xist developmental profile 17.

The quantitative accuracy of the PurAmp method was further confirmed by the fact that the Xist signal generated by a single blastomere isolated from a 4-cell female embryo arose 2.2 cycles later than the Xist signal of the whole 4-cell female embryo (compare light purple and red curves, Fig. 3). A left-to-right shift of 2 cycles is exactly what is expected for a fourfold decrease in template numbers quantified by real-time PCR amplification. Figure 4 illustrates this point by showing Xist RNA + genomic DNA levels in two individual blastomeres isolated from a 4-cell female embryo, as compared to Xist template levels measured in intact 4-cell embryos of different sex and then calculated on a per cell basis. Even at this early developmental stage, the presence of Xist RNA is clearly detectable in the female samples, absent from the male, and not affected by the blastomere isolation procedure 18.

In order to more extensively test the validity of the PurAmp approach to template quantification in single cells, we measured transcript levels of the heat shock-inducible genes hsp70.1 and hsp70.3 in blastomeres isolated from embryos at the pre-compaction 8-cell stage, when cells can be easily counted and separated. The sequences of these two genes are almost entirely identical, they are located on the same chromosome and they encode the same protein 1415. For this reason, there has been some confusion in their identification and nomenclature in past studies. Heat-inducible hsp70 transcription, previously indicated as hsp70.1 expression, is now more precisely designated as the sum of hsp70.1 and hsp70.3 (hsp70i) RNAs.

A preliminary set of experiments was carried out on embryos at the blastocyst stage, when heat shock response is fully established 14, with the goal of evaluating the effect of hyperthermia on hsp70i expression. Like Sry, the hsp70i are naturally intronless genes and therefore once again our pair of PCR primers simultaneously amplified both genomic DNA and cDNA sequences. The data in Table 1 clearly indicated that heat shock (see Methods) produced a sharp rise in hsp70i template numbers due to the presence of thousands of copies of hsp70i RNA, although these numbers were considerably lower when samples were prepared with a multistep/multitube phenol-chloroform extraction 1718 rather than with the PurAmp method. During these initial experiments, embryos were allowed to recover for 30–40 minutes after heat shock. Under these conditions, however, only five out of seven blastocysts exposed to hyperthermia showed an increase in hsp70i RNA levels. Based on these results, the duration of the recovery period was increased to at least two hours in all following experiments, eliminating the finding of "non responsive" embryos.

Copy numbers of hsp70i RNA were then quantified in whole 8-cell embryos that had or had not been exposed to a temperature increase, as summarized in Table 2. The number of hsp70i genomic DNA copies measured in the absence of RT was consistent with the presence of four copies of the genes per cell, one hsp70.1 and one hsp70.3 on each chromosome 17, and with the fact that some of the cells analyzed had already duplicated their DNA. Only a minimal amount of hsp70i RNA, calculated as the difference of template copy numbers obtained with and without reverse transcription (hsp70i cDNA + genomic DNA less hsp70i genomic DNA), was present in non-heated embryos, indicating that the embryos were not stressed by culture conditions 24.

Expression of hsp70i RNA increased sharply after a 30-minute heat treatment, followed by a recovery period of either 2 or 3 hours necessary for transcripts synthesis and accumulation. Some of the heat-shocked embryos were harvested intact, while others were dissociated in single blastomeres. The average numbers of hsp70i template copies per blastomere were calculated from the isolated cells (not all cells of dissected 8-cell embryos could be recovered) and compared to the average per blastomere values obtained from whole embryos. The results show that the amount of hsp70i RNA + genomic DNA per cell calculated by these two approaches is very similar. A post-heating recovery period of 3 hours rather than 2 hours increased the hsp70i RNA levels only slightly, indicating that the onset of transcription and the major build-up in RNA occur quickly. Single cells derived from non-heated embryos contained only trace amounts of hsp70i RNA, consistent with whole embryo measurements, and PCR efficiency was, again, unaffected by the PurAmp components (not shown).

The PurAmp method described above automatically results in the quantitative recovery of genomic DNA, which can then be used as a quality control and a convenient internal standard for the simultaneous recovery and measurement of mRNA [171819, see Discussion]. Applications such as microarray analysis, however, are based on RNA-only amplification. For this reason, we introduced a DNase digestion step preceding RT in the Xist/Sry PurAmp protocol and analyzed the efficacy with which the genomic DNA was degraded. Genome numbers in embryos at the morula stage were calculated by counting Xist and Sry copies in five male samples (as detailed for blastocysts, see above) and averaged at 21.3 ± 8.9, consistent with the fact that embryos at this stage are normally comprised of 16-to-32 cells. After treatment with DNase I, only 0.8 ± 1.5 genomes per embryo were still present in a group of 15 male samples, demonstrating that the enzyme had successfully degraded 96.3% of the DNA. Figure 5 illustrates the effects of DNase digestion on Xist (upper panel) and Sry (lower panel) DNA in a group of eight male and eight female single embryos. As expected, control male samples (blue lines) contained equal numbers of Xist and Sry copies, as shown by the equal C_T values, corresponding to the genomic DNA copy number. Both amplicons were absent from DNase-treated embryos that could be identified as male because they were devoid of Xist RNA (yellow lines). In contrast, control female samples (red lines) contained Xist RNA and DNA but lacked Sry. DNase treatment caused a delay in the Xist signals arising from female embryos (green lines), consistent with the elimination of all 42 copies of genomic Xist DNA in each embryo plus some decrease in the number of Xist transcripts. The amount of RNA recovered in these samples averaged at 75% of control levels. The fact that some RNA is lost during the DNase step is not surprising as it is well known that some RNA hydrolysis is unavoidable (see Discussion), and is not linked to the single-tube procedure. We anticipate that further optimization of the available DNase protocols and reagents will minimize this problem. Thus, the data in Fig. 5 demonstrate overall that a DNase digestion step can be successfully inserted within the PurAmp procedure, without disruption of the DNase enzymatic activity.

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 17 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 1819.

All experimental procedures were carried out using rigorous precautions aimed at avoiding or destroying environmental RNases contamination 171819.

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 39. 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 17. Dulbecco's PBS containing 0.4% polyvinyl pyrrolidone (both products from Sigma) was used when isolating single blastomeres 18. 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 39 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 171819, 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 40. 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) 1819. 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.

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) 14. 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 17. 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 17.

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 18. 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.

Calculation of template copy numbers was based on the "threshold cycle" (C_T) at which each fluorescent signal was first detected above background. C_T values were compared to standard scales obtained from analysis of male mouse genomes at known copy numbers, as detailed previously 171819 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 C_T determinations. A lower C_T value indicates an earlier detection of the fluorescent signal and therefore more templates present at the start of the reaction [reviewed in ref. 41]. 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).

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.