Semi-quantitative RT-PCR of mRNA derived from preimplantation bovine embryos

Figure 23: Schematic representation to determine the relative abundance of mRNA isolation

Digitalization by CCD camera

Quantification by densitometry

Digitalization by CCD camera

Quantification by densitometry

3.5.1 Isolation of mRNA

The relative abundance of transcriptional products of three developmentally important genes was analyzed in this experiment: The Insulin-like growth factor 2 gene (IGF2), the Insulin-like growth factor 2 receptor gene (IGF2R) and the Mammalian achaete-scute homologue 2 gene (MASH2). Single blastocysts, expanded and hatched blastocysts, ICM- and TE-cells from single expanded blastocysts (IGF2R, MASH2) and pools of nine expanded and hatched blastocysts (IGF2) were analyzed. mRNA was isolated using the Dynabeads® mRNA DIRECT Kit (Dynal, Oslo, Norway).

Embryos were stored at –80°C and kept in an ice block until 30 µl lysis buffer were added to the embryos. Immediately after adding lysis buffer to the embryos, 1 µl (1 pg) of rabbit globin mRNA was transferred to the embryo/lysis buffer solution, which was then centrifuged and incubated for 10 minutes at room temperature.

Rabbit globin mRNA served as an internal standard. Five µl of Dynabeads oligo (dT)25 were used to isolate mRNA from each sample. During embryo lysis, Dynabeads were washed twice in 20 µl lysis buffer which was discarded between the two washing steps using the magnetic separator. Five µl lysis buffer were finally added to the Dynabeads and transferred to the embryos. Binding of the Dynabeads to the polyA-tails of the embryonic mRNA was achieved in a thermo block at 26°C by shaking for 5 minutes. The tubes were transferred into the magnetic separator and the Dynabeads were fixed to the back of the tubes after 1 minute. The supernatant was discarded and the Dynabeads were washed once with 40 µl washing buffer A followed by three washing steps with 40 µl washing buffer B. Dynabeads were separated for 30 seconds before each washing step. Tubes were kept at room temperature outside of the magnetic separator after the last washing step and 11 µl of sterile water (22 µl when a negative control without RNase and Reverse Transcriptase was performed) were added to the Dynabeads. mRNA was eluted during incubation at 65°C for 2.5 minutes in a Biometra PCR cycler. The tubes were replaced into the ice-cold magnetic separator and mRNA was transferred to each RT reaction.

3.5.2 Reverse Transcription (RT)

Reverse transcription was performed in a final volume of 20 µl. Three different controls were included into the analyses:

1. Negative control containing embryonic mRNA, 1 pg rabbit globin mRNA and sterile water instead of RNase Inhibitor and Reverse Transcriptase to check the purity of the mRNA isolation and the absence of DNA contamination during RT-PCR. This type of negative control was done twice.

2. Negative control containing water instead of mRNA to ensure that samples were not contaminated.

3. Rabbit globin control with 1 pg globin mRNA, which was directly added to the RT reaction to estimate efficiency (it would have to be added to the RNA reaction to measure mRNA isolation efficiency).

The composition of the reaction mixture is shown in table 2. Reverse transcription was performed in a Biometra PCR cycler under the following conditions:

- 25°C for 10 minutes

- 42°C for 60 minutes (reverse transcription from mRNA into cDNA) - 99°C for 5 minutes (denaturation of extended enzymes)

- 4°C final temperature

Table 2: Composition of reaction mixture for reverse transcription

Component 1 x reaction mix Final concentration

10x PCR buffer⁴ 2 µl 1x

MgCl₂ (50 mM) 2 µl 5 mM

dNTP's (10 mM) 2 µl 1 mM

Hexamer primer⁵ (50 µM) 1 µl 2.5 µM

RNase inhibitor (20 U/µl) 1 µl 20 U

Reverse Transcriptase (50 U/µl) 1 µl 20 U

3.5.3 Gene specific PCR amplification

Primer pairs for analyses of the bovine IGF2R and MASH2 genes as well as for the rabbit globin gene were available from earlier experiments in the laboratory.

The primer pair for the bovine IGF2 gene was selected within the sequence of exon 10 (accession number: X53553). Table 5 shows details of the primer sequences, specific annealing temperatures and PCR cycle numbers.

Hot start PCR was performed in a final volume of 50 µl reaction mixture. The composition of the PCR mixture is described in table 3.

Table 3: Composition of the reaction mixture for gene specific PCR⁶

Component 1xreaction mix Final concentration

10x PCR buffer 4 µl 1x

MgCl2 (50 mM) 1.5 µl 1.5 mM

dNTP's (10 mM) 1 µl 200 µM

Primer (20 µM) 1.5 µl 0.6 µM

Sterile water to 50 µl

1 M Betain (only IGF2) 10 µl 0.2 M

DMSO (only MASH2) 3 µl 6%

Hot start mix

10xPCR buffer 1 µl

Sterile Water 10 µl

Taq polymerase (5 U/µl) 0.3 µl 1.5 U

PCR amplification of each gene specific transcript was performed in a hot lid PTC-200 thermocycler (MJ Research, Watertown, MA) under the conditions listed in table 4.

6 The amount of cDNA corresponding to embryonic equivalents is shown in table 5.

Table 4: PCR conditions for the amplification of gene specific transcripts

Gene transcript IGF2 IGF2R MASH2 Globin

Denaturation 95°C, 5 min 95°C, 2 min 95°C, 2 min 95°C, 2 min Adding hot start mix 72°C, 2 min 72°C, 2 min 72°C, 2 min 72°C, 2 min Denaturation 95°C, 20 sec 95°C, 15 sec 95°C, 15 sec 95°C, 15 sec Annealing 56°C, 30 sec 62°C, 15 sec 67°C, 15 sec 60°C, 15 sec Extension 72°C, 45 sec 72°C, 20 sec 72°C, 20 sec 72°C, 20 sec

Cycle number 39 33 37 27

Final extension 72°C, 10 min 72°C, 10 min 72°C, 10 min 72°C, 10 min

Final temperature 8°C 8°C 8°C 8°C

3.5.4 Electrophoresis and quantification of the relative amount of transcriptional products

Gene specific PCR products were subjected to electrophoresis on a 2%

agarose gel in 1 x TBE buffer. The gel was stained with 2 µl Ethidium Bromide (EtBr;

0.2 µg/ml). Separation of the PCR products was achieved through an initial 5 minutes at 100 V and 45 minutes at 80 V.

The PCR fragments were visualized on a 312 nm UV-transilluminator. The image of each gel was recorded with a CCD camera (Photometrics Inc.) and the IPLab Spectrum computer program (Signal Analytics Corporation). The camera had the ability to resolve 4,000 gray levels. The intensity of each gel band was detected by densitometry using the computer program IPLab Gel (Signal Analytics Corporation) and detection was based on the intensity of the gel bands in relation to the intensity of the background. Division of the intensity of each gel band by the intensity of the corresponding globin band resulted in determination of the relative amount of transcriptional products for each developmental stage and gene of interest. Between 5 and 9 replicates were performed for each embryonic stage and gene.

PCR products of the IGF2R and MASH2 genes were sequenced during previous experiments in our laboratory to verify the gene specific origin of the

Table 5: Details of primer pairs used for PCR amplification of gene specific embryonic mRNA

Genes Primer sequences^a

Annealing

a Sequences for forward (F) and reverse (R) primers are all written from 5' to 3' end.

b Primer pairs were designed from the sequence published in this reference and the PCR product was sequenced. This sequence was used to design the primer pairs shown in this table.

50 Materials and Methods

3.6 PCR amplification of CpG containing DNA regions in bovine tissue