Figure 4.

Poly(A) tail length regulation during oocyte maturation. (A) Schematic representation of the polyadenylation assay and poly(A) sequencing. mRNAs were ligated to a synthetic primer containing a 3′ amino blocking group (GB135). The ligation products are then used as templates for a reverse transcription reaction using GB136 primers complementary to GB135. To amplify cDNA and increase the specificity of the amplicons, first round amplification was performed using a gene-specific primer (P1) and GB136, and second round amplification was performed using a gene-specific primer which started after the 3′ site of P1 (P2) and GB136. The second round amplified product was then ligated with pGEMTM-T Easy plasmid, followed by transformation with JM109 cells before DNA sequencing. (B) Agarose gel electrophoresis of second round amplified products. Lane M: 100 bp ladder; Lane 1: positive control; Lanes 2–3: CCNB1 at germinal vesicle (GV) and metaphase II (MII) stage; Lanes 4–5: CCNB2 at GV and MII stage; Lanes 6–7: GAPDH at GV and MII stage; Lanes 8–9: HPRT1 at GV and MII stage. Uncropped blots are provided is Supplementary figure S2. DNA sequencing result for GAPDH at GV stage (C) and MII stage (D) aligned by cluster W method. The end sequence of GAPDH is outlined with the cyan box and sequence of GB135 is included in the orange box. (E) Length of poly(A) tail of GAPDH, CCNB1, CCNB2 and HPRT1 at the GV and MII stages. Significant differences are indicated by asterisks (*P < 0.05, ***P < 0.0005) and error bars represent standard deviation. Samples were collected from pools of 50 oocytes with three biological replicates.