Hello,

GlycoBlue is commonly used to aid in RNA precipitation and generally doesn't interfere with downstream applications. However, for mass spectrometry, it's essential to consider that GlycoBlue is a glycogen-based dye, and small residual amounts could potentially affect highly sensitive proteomic analyses due to additional signals or contaminants.

To minimize any interference, you could add a wash step after RNA precipitation to remove excess dye. Some protocols suggest washing the pellet with 70% ethanol a couple of times. This should help reduce any risk of GlycoBlue contamination in your mass spectrometry results.

Hello,

Thank you for your response! I always do EtOH washes. I hope it will work as expected.

GlycoBlue does not significantly affect mass spectrometry with RNA samples as it is primarily an inert glycogen-based co-precipitant used to enhance RNA recovery. However, its dye component might introduce minor background signals in highly sensitive analyses. To avoid potential interference, ensure thorough washing of RNA pellets. For critical applications, alternative co-precipitants like pure glycogen can be used.

Thank you everyone for the answer. It didn't affect the MS.

Using GlycoBlue to assist in RNA precipitation is a common practice, especially when working with minimal material. GlycoBlue is an inert co-precipitant that helps visualize the RNA pellet and enhances recovery. However, its use may raise some concerns about downstream applications.

Effects on Downstream Applications:

1. RT-PCR and qRT-PCR: GlycoBlue does not interfere with these applications as it is not enzymatically active and does not bind to RNA. Ensure thorough washing with ethanol during the precipitation process to remove residual salts or contaminants.

2. RNA-seq: GlycoBlue is RNA-free and should not interfere. However, any carryover should be minimal to avoid contamination in sensitive sequencing steps. Proper washing and handling will mitigate this.

3. Microarray Analysis: Similar to RNA-seq, GlycoBlue does not affect hybridization or labeling reactions if properly washed.

4. In vitro Translation: There is no known interference. As with other applications, residual contaminants from the precipitation process (e.g., salts or ethanol) should be minimized.

Key Considerations:

Amount Used: Use the recommended amount (typically 15–20 µg per sample) to avoid excessive GlycoBlue, which may complicate pellet washing.

Washing Steps: Proper washing with 70-80% ethanol is essential to remove any potential residues.

RNA Quality: Always check RNA integrity (e.g., using a bioanalyzer or gel electrophoresis) to ensure GlycoBlue did not alter the sample during the precipitation process.

In summary, GlycoBlue is highly compatible with most downstream applications, provided it is used correctly and washed thoroughly.