TNA Extraction Efficiency

The total nucleic acid (TNA) extraction might not have been successful or efficient enough to yield a high quantity of DNA and RNA in the sample. RNA may be present in higher quantities than DNA in the sample, which could explain why you see RNA bands but not TNA bands.

DNA Degradation

If the DNA was not effectively preserved during extraction or was degraded during handling (e.g., exposure to RNase contamination or suboptimal storage conditions), the TNA may contain very little or no DNA. In contrast, the RNA might be intact, which is why you see RNA bands.

Presence of RNase

RNase contamination in reagents or the workspace can degrade RNA, leaving only DNA in your sample. If this happens, you might only detect DNA if the TNA was extracted correctly. However, the absence of RNase inhibitors could lead to RNA degradation, resulting in no visible TNA bands.

RNA and DNA Separation

If your gel conditions were optimized for RNA separation, the DNA may be running as a smear, which might not be visible clearly. DNA typically migrates differently than RNA, so if there was a mix of RNA and DNA, the TNA may not resolve distinctly, especially if there’s too much RNA.

Gel Concentration and Staining

The concentration of the agarose gel may not be ideal for separating TNA or DNA from RNA. For larger DNA fragments, the agarose concentration might be too high. Additionally, ensure that you are using an appropriate DNA stain (e.g., ethidium bromide, SYBR Green) for TNA detection. It might also be a matter of under-staining or over-staining with the dye, affecting the visibility of the DNA.

Quantification of TNA

It's possible that the quantity of TNA was too low for visualization on the gel. You might want to check the concentration of your extracted TNA using a spectrophotometer (e.g., NanoDrop) to confirm that enough material was extracted for detection.

Buffer Composition

If the buffer used for running the gel wasn’t optimal for both RNA and DNA or if the electrophoresis conditions (e.g., voltage) weren't suitable for separating both types of nucleic acids, this could result in poor resolution of the TNA bands.