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What are the best practices and tips for RNA sample preparation in a high-throughput setting?

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RNA sample preparation is a crucial step for many high-throughput applications, such as RNA sequencing, microarrays, or qPCR. However, it can also be challenging and time-consuming, especially when dealing with large numbers of samples or complex biological materials. In this article, we will share some best practices and tips for RNA sample preparation in a high-throughput setting, covering topics such as sample collection, storage, homogenization, extraction, purification, quantification, and quality control.

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Verden Douglas

Quality Professional| Process Improvement

1 Sample collection and storage

The first step in RNA sample preparation is to collect and store your samples properly, as RNA is highly susceptible to degradation by RNases, heat, and pH changes. Ideally, you should process your samples as soon as possible after collection, or snap-freeze them in liquid nitrogen or dry ice and store them at -80°C until use. Avoid repeated freeze-thaw cycles, as they can damage the RNA integrity. If you are working with animal or human tissues, you may also need to add RNase inhibitors or stabilizers to prevent RNA degradation during transport or storage.

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Verden Douglas

Quality Professional| Process Improvement
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The starting material for the RNA extraction and purification must align with the extraction platform method. In other words, what is the required starting quantity for a successful product yield?

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2 Sample homogenization

The next step is to homogenize your samples, which means to disrupt the cells and release the RNA into a buffer. This can be done using mechanical methods, such as bead beating, grinding, or sonication, or chemical methods, such as lysis buffers or detergents. The choice of homogenization method depends on the type and size of your samples, the amount of RNA you need, and the downstream application. You should optimize the homogenization conditions to ensure efficient and consistent RNA extraction, while minimizing RNA degradation and contamination.

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3 RNA extraction and purification

After homogenizing your samples, you need to extract and purify the RNA from the buffer. There are different methods for RNA extraction and purification, such as phenol-chloroform extraction, spin column-based kits, or magnetic bead-based kits. Each method has its advantages and disadvantages, depending on the yield, purity, speed, cost, and scalability. You should select the method that best suits your sample type, RNA type, and downstream application. You should also follow the manufacturer's instructions carefully and avoid any sources of RNase contamination.

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4 RNA quantification and quality control

The final step in RNA sample preparation is to quantify and assess the quality of your RNA. This can be done using spectrophotometric methods, such as UV absorbance or fluorescence, or electrophoretic methods, such as agarose gel electrophoresis or capillary electrophoresis. Spectrophotometric methods can measure the concentration and purity of your RNA, while electrophoretic methods can evaluate the integrity and size distribution of your RNA. You should use both methods to ensure that your RNA meets the requirements of your downstream application, such as the minimum amount, the A260/A280 and A260/A230 ratios, and the RNA integrity number (RIN).

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5 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?

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What are the best practices and tips for RNA sample preparation in a high-throughput setting?

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1 Sample collection and storage

2 Sample homogenization

3 RNA extraction and purification

4 RNA quantification and quality control

5 Here’s what else to consider

Sample Preparation

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