Lecture Notes on MicroRNA and Detection Methods

Initial Sound Check and Overview

The session begins with a sound check, confirming that participants can hear clearly. There's an initial issue with screen sharing, ensuring the correct screen is being displayed to remote viewers.

Course Introduction and Review

The instructor addresses the participants, including those attending a make-up session. A recap of the previous week's material is planned, focusing on:

DNA

  • ATGC Base Pairing: The importance of Adenine (A), Thymine (T), Guanine (G), and Cytosine (C) base pairing in forming the double helix structure of DNA is emphasized.
    A−TA-T and G−CG-C represents the base pairing.
  • Central Dogma: A review of the central dogma of molecular biology is conducted, highlighting the flow of genetic information from DNA to RNA and then to protein. Protein synthesis is a later step.

Students who completed the homework assignment are acknowledged. Students in the make-up session are asked if they need to complete the homework as well.

Homework Review: MicroRNA and Disease

The homework involved researching 10 microRNAs (miRNAs) related to cardiovascular diseases (CVDs). The instructor notes the rapid completion of the assignment by many participants.

  • Expression Levels and Impact: The discussion covers how varying expression levels of miRNAs can affect disease outcomes. Upregulation or downregulation of miRNA expression can have beneficial or detrimental effects, depending on the specific miRNA and its target.
  • Example: MiR-21
    • Holleronit example: one example highlights the various targets of a microRNA. The instructor shows an example listing various microRNA targets.
    • Target Selection: Selecting the correct miRNA target is crucial to avoid unintended consequences. For example, targeting a gene involved in both cardiovascular disease and cancer could lead to adverse effects.
    • Multifactorial Considerations: Heart-related diseases are often related to many things that happen in the body.
    • Example Continued: If the goal is selecting for MiR-21 with cardiovascular disease, a highly expressed MiR-21 may also relate to cellular death or cancer cell growth; some patients who take chemotherapy show resistance to it because of MiR-21. The persuasiveness might not be enough, so if a result of high expression of MiR-21 comes up, it's unknown what the final outcome is.

Guidance on Analyzing MicroRNA Data

  • Data Interpretation: Students are advised to analyze expression data (e.g., from PAPPE, expression data tables) to determine whether a microRNA exhibits higher or lower expression in a particular context.
  • English vs. Translated Articles: Students are encouraged to read articles in English to better understand the nuances of the data.

Caution Regarding Overlapping Disease Associations

  • Multifaceted Roles of miRNAs: It's important to recognize that a single miRNA may be associated with multiple diseases.
  • Strategic Target Selection: When selecting a miRNA for therapeutic intervention, consider strategies to avoid potential off-target effects or unintended consequences.
    • Selecting multiple targets can help with accuracy, much like how doctors use several values to determine a disease.

Assay Development: Considerations for Target Selection

When designing an assay, focus on microRNAs that show consistent and specific expression patterns related to the target disease to increase the diagnostic accuracy.

  • Sample Source: The topic of the sample to test from arises, where blood is the easiest but not necessarily the most accurate.

Recap and Introduction to the Next Topic

The instructor summarizes the discussion and introduces the next topic: Recombinant DNA (rDNA).

  • Analide: Discussed in contrast to antibodies and enzymes.
  • Importance of Choosing the Right Approach: The instructor introduces the idea of why one would choose the correct form of Analide (nucleic acids, antibodies, or enzymes) and has the students discuss in groups.

Advantages of Nucleic Acid-Based Approaches

The discussion revolves around the benefits of using nucleic acids (DNA/RNA) in diagnostics and therapeutics.

  • Cost-Effectiveness: Nucleic acid-based approaches are generally cheaper than protein-based methods (e.g., antibodies).
  • Ease of Synthesis: The relative simplicity of nucleic acid sequences makes them easier and more cost-effective to synthesize on an industrial scale.
  • Programmability: Nucleic acids can be designed to bind to virtually any target sequence with high specificity, allowing for precise control over their activity.
    • Because there are only 4 possibilities of bases (A, T, G, C), they are easier to replicate for industrial purposes.
  • Base Pairing: DNA can bind to both DNA and RNA through base pairing. If BRNAs are AUCG, DNAPRO would be TAGC.

Discussion on Existing Detection Methods

The lecture transitions into a discussion comparing existing detection methods.

  • Existing Tools For Coding Test: Consideration of the existing tools for testing microRNA. Currently, these tools exist:
    • CGR
    • CEANCE -- lets the user test a lot from one place
    • MINCRAFRAY -- lets the user test a lot from one place
    • QPCR -- most commonly used in hospitals when C0DY was running rampant, but is not portable.
    • The first of these methods are old, where waiting a few days to receive results was normal.
  • Considerations:
    • Sensitivity: Some methods (e.g., QPCR) offer high sensitivity but require expensive equipment, specialized personnel, and are not easily adaptable for point-of-care applications.
    • Cost: Holge testing can be expensive at around 100k - 200k, with PCR costing around 10k. CUUS can cost less than 10k in order to stay in competition with ECR due to ECR's difficult nature.

Design of a Novel microRNA Detection System

The presentation shifts to a new system of detecting the microRNA by making it take the form of PAYPAN or a 3-strand DUCAS.

A new microRNA detection system is introduced, with a focus on overcoming the limitations of existing methods.

  • Key Design Elements:
    • AI Incorporation: Utilizing artificial intelligence (AI) to optimize the design and performance of the system.
    • Portability: Aiming for a compact, portable device that can be used in point-of-care settings or even with smartphones.
      • One design is about 5 cm and can hook up to a phone, with the potential to be more small and practical.
    • LANKER, ESTION and DEWPACE: Incorporating these into the DEWPACE shape, the LANKER and ESTION are bounded together in the PECHE sequence.
      • When the microRNA is found, the ACRANA acts as a DEWPACE where it slow moves to connect the microRNA.
      • At the STATTI0N, they become connected; this happens because of the STRUCTURD0K of red, blue and grey; the LANKER is also important in this.
      • The FILLEDSTEF0R squeezes out the LANKER into the 3-sided shape; it does not do anything to people but removes the various factors.
      • If there is no microRNA, the B cannot grab LANKER and will not emit it.