Dig rna ladder marker

Total RNA derived from the fat body of day 3 fifth instar larvae was used. Total RNA (12 μg) was separated on a 1.5% agarose, 6% formaldehyde gel and stained with ethidium bromide. Then, the gel was transferred to a nylon membrane. DIG-labeled probes were synthesized using the PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany) in accordance with supplier instructions using the following primers: BmSOD1, 5′-CACGAATTTGGTGACAACACAAATG-3′ and 5′-TTAAATCTTGGCCAAGCCAATGACT-3′; and BmSOD2, 5′-ATCAACTGTCGACAGCTTCTGT-3′ and 5′-TCACTTGAGCGCTTTTTCATA-3′. After pre-hybridization, membranes were hybridized with DIG-labeled probes at 50°C overnight. The specific reaction was visualized on Kodak XOMAT AR X-ray film using a DIG chemiluminescence detection kit (Roche Diagnostics). 18S ribosomal RNA (rRNA) was used as a control.
The size of the mRNA for both SODs was calculated using image analysis software CS Analyzer 3.0 (ATTO, Tokyo, Japan). A calibration curve was determined using the mobility of the DIG RNA ladder marker (Roche Diagnostics).

Total RNA derived from the testis of day 3 fifth-instar larvae was used. Total RNA (12 μg) was separated on a 1.5% agarose and 6% formaldehyde gel and stained with ethidium bromide. Next, the gel was transferred to a nylon membrane. DIG-labeled probes were synthesized using a PCR DIG probe synthesis kit (Roche Diagnostics, Mannheim, Germany) with specific primers (Table 3). After pre-hybridization, the membrane was hybridized with DIG-labeled probes at 50 °C overnight. The specific reaction was visualized on Kodak XOMAT AR X-ray film using a DIG chemiluminescence detection kit (Roche Diagnostics). 18S ribosomal RNA (rRNA) was used as a control. The mRNA size of BmEno genes was calculated using the image analysis software CS analyzer Ver. 3.0. A calibration curve was determined using the mobility of the DIG RNA ladder marker (Roche Diagnostics).